CN115144379B - Platelet ultrastructure imaging detection sample processing reagent, kit and application - Google Patents
Platelet ultrastructure imaging detection sample processing reagent, kit and applicationInfo
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Abstract
The invention discloses a platelet ultrastructural imaging detection sample processing reagent which comprises a diluent, wherein the ratio of a blood preservation solution to a desk type solution is 1:7-12. The invention also provides a kit for processing the platelet ultrastructural imaging detection sample, which comprises the sample processing reagent, and preferably further comprises a fixing solution, wherein the fixing solution comprises 4-8% by volume of paraformaldehyde, and a solvent comprises PHEM buffer with pH of 6.8-7.2, and the fixing solution of the platelet microtubule structure preferably comprises 5.5-6.6% by volume of paraformaldehyde, 0.05-0.15% by volume of glutaraldehyde and 0.1-0.2% by volume of TritonX 100. In addition, the invention also provides an application of the platelet ultrastructural imaging detection sample treatment reagent in preparing an imaging detection sample, diluent is added after platelet plasma obtained by centrifugation, and platelets are fixed again after dilution and standing, so that the non-activated platelets can be obtained, and the detection result is better close to the real condition of the platelets under physiological conditions.
Description
Technical Field
The invention belongs to the technical field of platelet structure detection, and particularly relates to a platelet ultrastructural imaging detection sample processing reagent, a kit and application.
Background
The super-resolution fluorescence microscopic imaging technology breaks through the limit of optical diffraction, realizes the nano-scale optical detection in cells, provides a brand-new means for detecting subcellular structures (such as microfilaments, microtubules, mitochondria and the like) in cells, and has very few application in the field of platelet detection.
Platelets are a very sensitive and easily activated cell type compared to normal cells, and the subcellular structure of the activated platelets is changed, so that the platelets under study are difficult to represent the real state of circulating platelets under physiological conditions. Therefore, imaging of the ultrastructural structure of platelets needs to be performed without activating the platelets, which puts high demands on experimental operations, which is often difficult to achieve with existing detection means.
At present, the super-resolution fluorescence imaging of the ultrastructure of the blood platelet is only aimed at fixing dead cells, but before the blood platelet is fixed, the blood platelet is required to be separated and purified from whole blood, the activation of the blood platelet is easy to cause in the process, the reason is not detailed, and the problem is not reported in the related solution at present.
Disclosure of Invention
Aiming at the defects or improvement demands of the prior art, the invention provides a platelet ultrastructural imaging detection sample processing reagent, a kit and application thereof, and aims to dilute a diluent prepared by adding blood preservation solution and desk-top liquid and a centrifuged plasma sample, so as to remarkably reduce the concentration of an anticoagulant in the centrifuged plasma, thereby obtaining unactivated platelets, and solving the technical problems that the platelets obtained by the existing separation and purification are usually activated platelets and are difficult to represent the real state of circulating platelets under physiological conditions.
In order to achieve the above object, according to one aspect of the present invention, there is provided a sample processing reagent for imaging and detecting a platelet ultrastructural structure, wherein the sample processing reagent comprises a diluent, and the diluent comprises a blood preservation solution and a desk top solution, wherein the volume ratio of the blood preservation solution to the desk top solution is 1:7-12.
Preferably, the ratio of the blood preservation solution to the desk top solution of the diluent is 1:9.
Preferably, the table fluid comprises 7.5-8.5 g/L sodium chloride, 0.18-0.25 g/L potassium chloride, 0.18-0.25 g/L magnesium chloride hexahydrate, 0.58-0.72 g/L sodium dihydrogen phosphate dihydrate, 0.90-1.15 g/L sodium bicarbonate, 0.85-1.05 g/L glucose and 2.15-2.50 g/L4-hydroxyethyl piperazine ethane sulfonic acid, wherein the solvent is water, and the pH is 7.0-7.5.
According to another aspect of the invention there is also provided the use of a reagent according to the invention for the preparation of a sample for the imaging detection of ultrastructural platelets.
Preferably, the application of the platelet ultrastructural imaging detection sample processing reagent in preparing an imaging detection sample comprises the steps of firstly diluting the platelet sample mixed with the anticoagulant after centrifugation, then fixing and marking to obtain the detection sample to be imaged, wherein the platelet sample is diluted and protected according to the volume ratio of the platelet sample after centrifugation to the diluent of 1:4-10, and the anticoagulant comprises EDTA salt type anticoagulant.
Preferably, the use of the platelet ultrastructural imaging test sample processing reagent for preparing an imaging test sample comprises the steps of:
(1) The whole blood centrifugal separation to obtain platelet plasma, namely, a blood collection tube is used for obtaining whole blood, and the platelet-rich plasma is obtained through centrifugation, wherein EDTA salt type anticoagulant is mixed in the platelet-rich plasma;
(2) Platelet pre-protection treatment, namely diluting the platelet plasma obtained in the step (1) with the diluent according to the volume ratio of 1:4-10, and standing for recovery;
(3) Platelet fixation, namely adding a fixing solution into the platelets after standing and recovering in the step (2), fixing, centrifuging to remove supernatant and keep platelet sediment, centrifuging after the platelet sediment is resuspended by phosphate buffer solution, removing supernatant, and resuspending the fixed platelets by adopting phosphate buffer solution to obtain platelet suspension;
(4) Preparing a detection sample, namely taking the platelet suspension in the step (3) according to the condition of sample platelet counting, spreading the platelet suspension in a culture dish after dilution, standing and settling for more than 1 hour, and obtaining the detection sample to be imaged by platelet immunofluorescence staining and marking.
Preferably, the application of the platelet ultrastructural imaging detection sample treatment reagent in preparing an imaging detection sample is characterized in that the fixing solution in the step (3) comprises 5.5% -6.6% of paraformaldehyde, 0.05% -0.15% of glutaraldehyde and 0.1% -0.2% of Triton X-100 for the platelet microtubule structure, and the solvent comprises PHEM buffer solution with pH of 6.8-7.2.
According to another aspect of the present invention, there is also provided a kit for processing a platelet ultrastructural imaging test sample, the kit comprising a platelet ultrastructural imaging test sample processing reagent according to the present invention.
The kit for processing the platelet ultrastructural imaging detection sample comprises a fixing solution and a marking reagent, wherein the fixing solution comprises 4-8% by volume of paraformaldehyde, the solvent comprises PHEM buffer with pH of 6.8-7.2, and the marking reagent comprises primary anti-dilution solution and fluorescent secondary anti-dilution solution for marking one or more of platelet microtubules, compact particles, mitochondria and alpha particles.
Preferably, the fixing solution of the kit for processing the platelet ultrastructural imaging detection sample comprises 5.5% -6.6% of paraformaldehyde, 0.05% -0.15% of glutaraldehyde and 0.1% -0.2% of Triton X-100 by volume.
In general, the above technical solutions conceived by the present invention, compared with the prior art, enable the following beneficial effects to be obtained:
The platelet ultrastructural imaging detection sample processing reagent provided by the invention comprises a diluent, wherein the diluent comprises a blood preservation solution and a desk type solution, the diluent and the desk type solution are proportioned to reduce the probability of activating platelets separated by a centrifugal method in a standing recovery process, the volume ratio of the blood preservation solution to the desk type solution is preferably 1:7-12, and the diluent under the proportioned condition can well protect the platelets from being activated, so that the detection result of the platelet ultrastructural detection is closer to the in-vivo condition. Therefore, the platelet ultrastructural imaging detection sample processing reagent provided by the invention can obtain a platelet to-be-detected sample which is not activated.
The invention also provides application of the platelet ultrastructural imaging detection sample treatment reagent in preparation of imaging detection samples, the platelet ultrastructural imaging detection sample treatment reagent is improved on the basis of the existing method, the platelet ultrastructural imaging detection sample treatment reagent is added with diluent for dilution after centrifugation to obtain platelet plasma, then the platelet is kept still for recovery, platelets are fixed after the standing recovery, the platelet ultrastructural structure is detected after immunofluorescent staining, the platelet is diluted by adding the diluent of the invention after centrifugation, the activated number of the platelets can be obviously reduced, the platelet ultrastructural imaging detection sample treatment reagent can be detected under the condition that the platelets are not activated, and the detection result is closer to the real state of circulating platelets under physiological conditions, so that the platelet ultrastructural imaging detection reagent has great significance for medicine or disease research.
In addition, the sample processing kit for the imaging detection of the platelet ultrastructure comprises the sample processing reagent, and preferably further comprises fixing solution, wherein the fixing solution for the platelet microtubule structure comprises paraformaldehyde, glutaraldehyde (GA) and Triton X-100, and the fixing solution, the glutaraldehyde (PFA), the Glutaraldehyde (GA) and the microtubule fixing solution are used cooperatively to reduce the change of the fixing solution reagent to the platelet microtubule structure, and particularly comprise 5.5-6.6% of Paraformaldehyde (PFA), 0.05-0.15% of Glutaraldehyde (GA) and 0.1-0.2% of microtubule fixing solution of Triton X-100, and the microtubule structure of the platelet is hardly changed after the platelet is fixed. The kit provided by the invention has wide application prospect.
Drawings
FIG. 1 is a photograph of a platelet microtubule;
FIG. 2 is a photograph of dense particles of platelets;
FIG. 3 is a photograph of platelet mitochondria;
FIG. 4 is a photograph of platelet alpha particles;
FIG. 5 is a comparison of microtube structures taken after preparing samples with 3 sample processing reagents (dilutions differ);
FIG. 6 is a graph comparing microtube structures taken after preparing samples using 3 sample processing reagents (fixative solutions).
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
Platelets obtained by a centrifugal separation method in a blood sample contained in a conventional blood collection tube (purple head tube) in a hospital show changes in a plurality of microtubule structures in subsequent super-resolution imaging, but the platelets obtained by gel column separation rarely show the changes, and the analysis reasons probably are that the platelets obtained by centrifugal separation are activated much and the platelets obtained by gel column separation are activated rarely. Further analysis of these two methods has found that, after centrifugation, a long-time stationary recovery treatment is required for the plasma sample mixed with EDTA salt type anticoagulants, and the concentration of the anticoagulants in the purple head blood collection tube is high and coexists with platelets for a long time during stationary recovery, so that it is presumed that the activation of platelets is caused by the coexistence of the anticoagulants with high concentration and platelets for a long time.
Compared with the centrifugal separation method, the method for gel separation has the advantages that the number of the obtained platelets is too small, the collection and statistics of subsequent data of imaging detection are inconvenient, so that the centrifugal separation method is better when the large-scale super-resolution imaging is carried out, detection and statistics are carried out, but when the sample is processed by adopting centrifugal separation, the platelets to be detected are activated, so that the detection result is greatly different from the physiological state of circulating platelets under the physiological condition, and further analysis shows that the platelets are activated due to high-concentration anticoagulants through our experiments.
Therefore, the invention provides that the platelet plasma obtained after centrifugal separation is added with diluent, then standing recovery and super-resolution imaging detection are carried out, and experimental results prove that the phenomenon that the platelets are activated during the detection of the sample to be detected obtained by the method is obviously reduced.
The invention provides a platelet ultrastructural imaging detection sample processing reagent, which comprises a diluent, wherein the diluent comprises a blood preservation solution and a desk type solution, and the volume ratio of the blood preservation solution to the desk type solution is preferably 1:7-12, and the diluent under the ratio can better protect platelets from being activated, and the diluent with the volume ratio of the blood preservation solution to the desk type solution being more preferably 1:9;
The desk top liquid comprises 7.5-8.5 g/L of sodium chloride (NaCl), 0.18-0.25 g/L of potassium chloride (KCl), 0.18-0.25 g/L of magnesium chloride hexahydrate (MgCl 2·6H2 O), 0.58-0.72 g/L of sodium dihydrogen phosphate dihydrate (NaH 2PO4·2H2 O), 0.90-1.15 g/L of sodium bicarbonate (NaHCO 3), 0.85-1.05 g/L of glucose, 2.15-2.50 g/L of 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES), water as a solvent and pH of 7.0-7.5.
The blood preservation solution refers to citric acid-sodium citrate-glucose (ACD solution for short), and the pH value is between 4.5 and 5.5, such as blood preservation solution (I), blood preservation solution II and blood preservation solution III.
In addition, the invention also provides application of the platelet ultrastructural imaging detection sample processing reagent in preparation of platelet ultrastructural imaging detection samples.
The application comprises the following steps:
(1) The whole blood is centrifugally separated to obtain platelet plasma, namely a blood collecting tube (a purple head tube) is used for obtaining whole blood, and the platelet-rich plasma is centrifugally obtained, wherein EDTA salt type anticoagulants with higher concentration, such as EDTA-K2/EDTA-K3 anticoagulants, are mixed in the platelet-rich plasma;
(2) Platelet pre-protection treatment, namely diluting the platelet plasma obtained in the step (1) with a diluent according to a preset proportion, and standing for recovery, wherein the preset proportion is preferably that the platelet plasma is diluent=1:4-10;
The experiment result shows that the phenomenon that the blood preservation solution (ACD) or the desk type solution is singly used as the dilution solution is not obviously reduced, the blood preservation solution possibly contains sterilized aqueous solution of sodium citrate, citric acid and glucose, the pH value of the solution is 4.5-5.5, the acidic dilution solution easily causes the activation of the blood platelets, the desk type solution has the functions of maintaining osmotic pressure, controlling acid-base balance and supplying energy and inorganic salt necessary for cell metabolism survival, but is singly used as the dilution solution, the blood platelets are activated, obvious dispersion phenomenon occurs in a microtubule structure, the blood preservation solution and the desk type solution are matched for use, the dispersion phenomenon of the microtubule structure of the blood platelets is very small, the protection effect on the blood platelets is obviously superior to that of the blood preservation solution and the desk type solution, the volume ratio of the blood preservation solution to the desk type solution is preferably 1:7-12, the dilution solution under the ratio can better protect the blood platelets from being activated, and the very close to the real state is provided for researching the ultra-micro structure of the blood platelets.
(3) Platelet fixation, namely adding a fixation solution with the same volume as the diluent in the step (2) into the platelets after standing recovery obtained in the step (2), fixing, centrifuging to remove supernatant and keep platelet sediment to obtain the platelets after fixation, re-suspending the sediment by using Phosphate Buffer (PBS), centrifuging to remove supernatant, and re-suspending the platelets after fixation by using PBS solution to obtain platelet suspension.
The fixing solution of the platelet microtubule structure adopts paraformaldehyde PFA, glutaraldehyde (GA) and Triton X-100 to be matched, because the microtubule structure of the platelet is a very sensitive and easy-to-change structure, the structure shot after the fixation of the platelet microtubule structure is singly adopted by paraformaldehyde PFA is easy to change, the fixation of the platelet microtubule structure by adopting PFA, glutaraldehyde (GA) and Triton X-100 has small influence on the microtubule structure change, and preferably comprises 5.5-6.6% of Paraformaldehyde (PFA), 0.05-0.15% of Glutaraldehyde (GA) and 0.1-0.2% of Triton X-100, under the matching condition, the imaging structure of the microtubule after the fixation is clearer, the microtubule structure is hardly influenced, and the fixation of other common fixing reagents can lead to obvious dispersion of the microtubule structure.
(4) Preparing a detection sample, namely taking the platelet suspension in the step (3), diluting, spreading the diluted platelet solution into a glass bottom culture dish, standing and settling for more than 1 hour, and carrying out immunofluorescence staining on the platelets to obtain an immunofluorescence-stained sample to be detected.
When the sample to be tested is used for detecting the ultrastructure of the blood platelet, the ultrastructure of the blood platelet is detected by adopting the super-resolution fluorescence imaging, and the change information of the ultrastructure of the blood platelet can be obtained through analysis and statistics of the morphological change of the ultrastructure of the blood platelet.
The invention also provides a platelet ultrastructural imaging detection sample processing kit, which comprises the platelet ultrastructural imaging detection sample processing reagent, preferably a fixing solution;
The fixing solution comprises 4-8% by volume of Paraformaldehyde (PFA), and the solvent comprises PHEM buffer solution, wherein the pH of the PHEM buffer solution is 6.8-7.2, and the fixing solution for the platelet microtubule structure further comprises Glutaraldehyde (GA) and Triton X-100, preferably comprises 5.5-6.6% by volume of Paraformaldehyde (PFA), 0.05-0.15% by volume of Glutaraldehyde (GA) and 0.1-0.2% by volume of Triton X-100. The imaging structure after the microtube is fixed under the condition is clearer, the fixation of other common fixing reagents can lead the structure of the microtube to be obviously dispersed, and the fixing liquid of the platelet microtube structure more preferably comprises 6 volume percent of Paraformaldehyde (PFA), 0.1 volume percent of Glutaraldehyde (GA) and 0.6 volume percent of Triton X-100, and the solvent comprises PHEM buffer solution.
The PHEM buffer solution preferably comprises 17.5-18.5 g/L1, 4-piperazine diethyl sulfonic acid (PIPES), 4.8-7.2 g/L4-hydroxyethyl piperazine ethane sulfonic acid (HEPES), 2.2-3.6 g/L ethylene glycol-bis (2-aminoethyl) tetraacetic acid (EGTA), 0.35-0.55 g/L magnesium chloride hexahydrate (MgCl 2.6H2 O), water as a solvent and pH of 6.8-7.2.
The kit more preferably further comprises a labeling reagent, wherein the labeling reagent comprises one or more of primary anti-diluent and fluorescent secondary anti-diluent of cell perforating liquid, blocking liquid, cleaning liquid, labeled platelet microtubule, compact particle, mitochondria and alpha particle.
The following are examples:
example 1 platelet ultrastructural (microtubule structure) sample processing reagent 1
The platelet ultrastructural imaging detection sample treatment reagent 1 comprises a diluent, a fixing solution and a marking reagent, wherein the diluent comprises 10% of blood preservation solution (I) and 90% of desk top solution, wherein the desk top solution comprises 8.064g/L of sodium chloride (NaCl), 0.216g/L of potassium chloride (KCl), 0.203g/L of magnesium chloride hexahydrate (MgCl 2·6H2 O), 0.6552g/L of sodium dihydrogen phosphate dihydrate (NaH 2PO4·2H2 O), 1.0092g/L of sodium bicarbonate (NaHCO 3), 0.9908g/L of glucose and 2.384g/L of 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES), and the solvent is water with the pH of 7.2;
The fixing solution comprises 6% of Paraformaldehyde (PFA), 0.1% of Glutaraldehyde (GA) and 0.15% of Triton X-100 by volume, wherein the solvent is PHEM buffer solution, and the PHEM buffer solution comprises 18.147g/L of 1, 4-piperazine diethyl sulfonic acid (PIPES), 5.956g/L of 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES), 2.922g/L of glycol-bis- (2-aminoethyl) tetraacetic acid (EGTA), 0.406g/L of magnesium chloride hexahydrate (MgCl 2·6H2 O), and the solvent is water with pH of 7.0;
The labeling reagent comprises a cell punching liquid, a blocking liquid, a cleaning liquid, a primary anti-dilution solution and a fluorescent secondary anti-dilution solution, wherein the cell punching liquid is a PBS solution containing 0.2% Triton X-100, the blocking liquid is a PBS solution containing 10% goat serum (NAS), 1% Bovine Serum Albumin (BSA) and 0.05%Triton X~100,0.05% antibacterial preservative Proclean, the cleaning liquid is a PBS solution containing 0.1% Tween20, the primary and secondary anti-dilution solutions of the platelet microtubules are a murine alpha-tubulin monoclonal antibody (primary antibody) diluted in the blocking liquid by 1:500, and an anti-murine Alexa Fluor 488 antibody (secondary antibody) is diluted in the blocking liquid by 1:500.
Example 2 platelet ultrastructural (dense particle) sample processing reagent 2
The platelet ultrastructural imaging detection sample processing reagent 2 comprises a diluent, a fixing solution and a marking reagent, wherein the volume ratio of a blood preservation solution to a desk top liquid of the diluent is 1:9, the fixing solution comprises 8% by volume of Paraformaldehyde (PFA), and a solvent is PHEM buffer (same as in example 1);
the labeling reagent comprises a cell perforating liquid, a sealing liquid and a cleaning liquid (same as in the embodiment 1), and a primary anti-dilution solution and a fluorescent secondary anti-dilution solution for labeling compact particles;
Wherein the diluted solution of the compact particles of the primary antibody and the secondary antibody is a murine CD63 monoclonal antibody (primary antibody) diluted 1:100 in a blocking solution, and the anti-murine Alexa Fluor 488 antibody (secondary antibody) diluted 1:500 in the blocking solution.
Example 3 platelet ultrastructural (mitochondrial) sample processing reagent 3
The platelet ultrastructural imaging detection sample processing reagent 3 comprises a diluent, a fixing solution and a marking reagent, wherein the volume ratio of a blood preservation solution to a desk top solution of the diluent is 1:7, the fixing solution comprises 8% by volume of Paraformaldehyde (PFA), and a solvent is PHEM buffer (same as in example 1);
The labeling reagent comprises a cell perforating liquid, a sealing liquid and a cleaning liquid (same as in the example 1), and a primary anti-dilution solution and a fluorescent secondary anti-dilution solution for labeling mitochondria;
Wherein the mitochondrial primary and secondary antibodies are rabbit TOMM20 monoclonal antibody (primary antibody) diluted 1:250 in blocking solution, and anti-rabbit Alexa Fluor 488 antibody (secondary antibody) diluted 1:500 in blocking solution.
Example 4 platelet ultrastructural (alpha particles) sample processing reagent 4
The platelet ultrastructural imaging detection sample processing reagent 4 comprises a diluent, a fixing solution and a marking reagent, wherein the volume ratio of a blood preservation solution to a desk top solution of the diluent is 1:12, the fixing solution comprises 8% by volume of Paraformaldehyde (PFA), and a solvent is PHEM buffer (same as in example 1);
the labeling reagent comprises a cell perforating liquid, a sealing liquid and a cleaning liquid (same as in the embodiment 1), and a primary anti-dilution solution and a fluorescent secondary anti-dilution solution for labeling alpha particles;
Wherein the primary and secondary antibody dilution solutions of the alpha particle antibody are rabbit VWF monoclonal antibodies (primary antibodies) diluted 1:1000 in the blocking solution, and the anti-rabbit Alexa Fluor 488 antibodies (secondary antibodies) diluted 1:500 in the blocking solution.
Example 5 platelet ultrastructural detection
After sample processing reagents in examples 1 to 4 are adopted to prepare detection samples according to the processing method provided by the invention, the detection is carried out by utilizing the super-resolution fluorescence imaging technology, and the method specifically comprises the following steps:
(1) Whole blood separation to obtain platelets Whole blood obtained from blood collection tubes was centrifuged at 200g for 12 minutes to obtain platelet rich plasma.
(2) Platelet pre-protection treatment, namely diluting platelet-rich plasma and a diluent in a ratio of 1:4-10, and then placing the diluted platelet-rich plasma and the diluent in a 37 ℃ and 5% CO 2 incubator for standing and recovering for two hours.
(3) Platelet immobilization, namely adding an equal volume of immobilization solution into the platelet suspension after standing and recovering, standing and fixing for 30 minutes, centrifuging 1500g for 3 minutes, removing supernatant to retain platelet sediment, re-suspending the sediment with 1mL of PBS solution, centrifuging 1500g for 3 minutes again, removing supernatant, and re-suspending the immobilized platelets with 5mLPBS solution.
(4) Platelet spreading dish, namely diluting a proper amount of platelet suspension according to the counting condition of sample platelets, spreading 200 mu L of diluted platelet solution into a glass bottom culture dish, and standing and settling for at least 1 hour.
(5) Platelet immunofluorescence staining platelet punching by adding 200uL of cell punching solution to the dish, standing for 10min, and discarding the solution. Platelet was blocked, 200uL of blocking solution was added to the dish and allowed to stand for 1 hour, after which the solution was discarded. And (3) performing primary antibody incubation, namely adding 150uL primary anti-dilution solution into a dish, performing co-incubation of the antibody and platelets at 4 ℃ for 12-16 h, discarding liquid after incubation, and washing with a cleaning liquid for 5 times each time for 5 minutes. And (3) secondary antibody incubation, namely 150uL of secondary antibody dilution solution is added into a dish, the antibody and the platelet are incubated for 1 hour at room temperature, the liquid is discarded after incubation, and the dish is washed with a cleaning liquid for 5 times and 5 minutes each time. Post-treatment 200ul 4% Paraformaldehyde (PFA) was added to the dish for fixation for 10 min. After fixation, the liquid was discarded, and after washing 3 times with PBS, the dishes were kept at 4 ℃.
The above sample after sample preparation is subjected to super-resolution fluorescence imaging detection, and the results are shown in fig. 1-4, wherein fig. 1 is a shooting result of a platelet microtubule, fig. 2 is a shooting result of a platelet dense particle, fig. 3 is a shooting result of a platelet mitochondria, and fig. 4 is a shooting result of a platelet alpha particle.
Comparative example 1 platelet ultrastructural (microtubule structure) sample processing reagent 5
The platelet ultrastructural imaging detection sample processing reagent comprises a diluent, a fixing solution and a marking reagent, wherein the diluent is 100% blood preservation solution (ACD), and the fixing solution is the same as that of the embodiment 1.
Comparative example 2 platelet ultrastructural (microtubule structure) sample processing reagent 6
The platelet ultrastructural imaging detection sample processing reagent comprises a diluent, a fixing solution and a marking reagent, wherein the diluent is 100% desk type liquid, and the fixing solution is the same as that in the embodiment 1.
Since the structure of microtubules changes most significantly inside platelets, the test results are more representative, and the following samples are tested for microtubule structure after treatment, which is as follows:
Using the sample processing reagents of example 1, comparative example 1 and comparative example 2, a test sample was prepared according to the processing method provided by the present invention (same as example 5), and the test was performed using the super-resolution fluorescence imaging technique, and the result is shown in fig. 5.
Comparative example 3 platelet ultrastructural (microtubule structure) sample processing reagent 7
The platelet ultrastructural imaging detection sample processing reagent 7 comprises a diluent, a fixing solution and a marking reagent, wherein the ratio of a blood preservation solution to a desk top solution of the diluent is 1:9, the fixing solution is 8% Paraformaldehyde (PFA), and the solvent is PHEM buffer (same as in example 1).
Comparative example 4 platelet ultrastructural (microtubule structure) sample processing reagent 8
The platelet ultrastructural imaging detection sample processing reagent 7 comprises a diluent, a fixing solution and a marking reagent, wherein the ratio of the diluent to a blood preservation solution and a desk top solution is 1:9, the fixing solution is 4% Glutaraldehyde (GA), and the solvent is PHEM buffer solution (same as in example 1).
Using the sample processing reagents of example 1, comparative example 3 and comparative example 4, a test sample was prepared according to the processing method provided in the present invention (same as example 5), and living cells were used as a control, and the result was shown in fig. 6 by using the super-resolution fluorescence imaging technique.
As can be seen from FIG. 6, the platelet microtubule structure was dispersed by using 8% or 4% glutaraldehyde as the fixing solution, wherein the dispersion was more pronounced after the treatment with the reagent of comparative example 4, whereas the sample was treated with the sample treatment reagent of the present invention, which showed no significant difference from the platelet microtubule structure in living cells, indicating that the platelets were hardly activated when the sample was treated with the sample treatment reagent provided by the present invention.
It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the invention and is not intended to limit the invention, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (6)
1. The sample processing reagent for the platelet ultrastructural imaging detection is characterized by comprising a diluent and a fixing solution for inhibiting platelet from being activated, wherein the diluent comprises a blood preservation solution and a desk type solution, and the pH value of the blood preservation solution is 4.5-5.5;
when the platelet ultrastructure is a microtube, the volume ratio of the blood preservation solution to the desk top solution in the diluent is 1:9, the fixing solution comprises 5.5-6.6% of paraformaldehyde, 0.05-0.15% of glutaraldehyde and 0.1-0.2% of Triton X-100 by volume percent, and the solvent comprises PHEM buffer solution with pH of 6.8-7.2;
When the platelet ultrastructure is compact particles, the volume ratio of the blood preservation solution to the desk type solution in the diluent is 1:9, the fixing solution comprises 4-8% paraformaldehyde by volume percent, and the solvent comprises PHEM buffer solution with pH of 6.8-7.2;
When the platelet ultrastructure is mitochondria, the volume ratio of the blood preservation solution to the desk type solution in the diluent is 1:7, the fixing solution comprises 4-8% paraformaldehyde by volume percent, and the solvent comprises PHEM buffer solution with pH of 6.8-7.2;
When the platelet ultrastructure is alpha particles, the volume ratio of the blood preservation solution to the desk top liquid in the diluent is 1:12, the fixing solution comprises 4-8% of paraformaldehyde by volume percent, and the solvent comprises PHEM buffer solution with pH of 6.8-7.2.
2. The reagent for processing a platelet ultrastructural imaging test sample according to claim 1, wherein the tabletop liquid comprises 7.5 to 8.5g/L sodium chloride, 0.18 to 0.25g/L potassium chloride, 0.18 to 0.25g/L magnesium chloride hexahydrate, 0.58 to 0.72g/L sodium dihydrogen phosphate dihydrate, 0.90 to 1.15g/L sodium bicarbonate, 0.85 to 1.05g/L glucose, and 2.15 to 2.50 g/L4-hydroxyethyl piperazine ethane sulfonic acid, and the solvent is water, and the pH is 7.0 to 7.5.
3. Use of a platelet ultrastructural imaging test sample processing reagent according to any one of claims 1 or 2 for preparing a platelet ultrastructural imaging test sample, characterized in that the platelet sample mixed with anticoagulant obtained by centrifugation is diluted with a diluent and then fixed with a fixing liquid.
4. The application of the reagent for treating the platelet ultrastructural imaging detection sample, according to the application, the platelet sample mixed with the anticoagulant after centrifugation is diluted by a diluent, and is fixed by a fixing solution after standing and recovering, and the sample to be imaged is obtained by marking, wherein the diluent is used for diluting and protecting the platelet according to the volume ratio of the platelet sample after centrifugation to the diluent of 1:4-10, and the anticoagulant comprises EDTA salt type anticoagulants;
when the platelet ultrastructure is a microtube, the volume ratio of the blood preservation solution to the desk top solution in the diluent is 1:9, the fixing solution comprises 5.5-6.6% of paraformaldehyde, 0.05-0.15% of glutaraldehyde and 0.1-0.2% of Triton X-100 by volume percent, and the solvent comprises PHEM buffer solution with pH of 6.8-7.2;
When the platelet ultrastructure is compact particles, the volume ratio of the blood preservation solution to the desk type solution in the diluent is 1:9, the fixing solution comprises 4-8% paraformaldehyde by volume percent, and the solvent comprises PHEM buffer solution with pH of 6.8-7.2;
When the platelet ultrastructure is mitochondria, the volume ratio of the blood preservation solution to the desk type solution in the diluent is 1:7, the fixing solution comprises 4-8% paraformaldehyde by volume percent, and the solvent comprises PHEM buffer solution with pH of 6.8-7.2;
When the platelet ultrastructure is alpha particles, the volume ratio of the blood preservation solution to the desk top liquid in the diluent is 1:12, the fixing solution comprises 4-8% of paraformaldehyde by volume percent, and the solvent comprises PHEM buffer solution with pH of 6.8-7.2.
5. A kit for the imaging detection of a platelet ultrastructural sample treatment, characterized in that it comprises a platelet ultrastructural imaging detection sample treatment reagent according to any one of claims 1 or 2.
6. The kit for imaging detection sample processing of platelet ultrastructural system according to claim 5, further comprising a labeling reagent comprising a primary anti-dilution solution and a fluorescent secondary anti-dilution solution for labeling one or more of platelet microtubes, dense particles, mitochondria, alpha particles.
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| Superresolution Fluorescence Microscopy of Platelet Subcellular Structures as a Potential Tumor Liquid Biopsy;Xu Peng等;SMALL METHODS;20230622;第7卷(第10期);2300445 * |
| 三七总皂苷基于花生四烯酸代谢通路保护胃黏膜和增强阿司匹林抗血小板作用的实验研究;王文婷等;中西医结合心脑血管病杂志;20190510;第17卷(第09期);第1315-1320页 * |
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