The role of fixation, from the histological point of view its simple definition is to maintain the intrinsic morphology and structure of cells and tissues, so that as far as possible to maintain the intrinsic morphology and structure of cells and tissues, and from the point of view of immunohistochemistry technology, the role of fixation not only to make intracellular proteins coagulation, minimize or terminate the reaction of exogenous enzymes and endogenous enzymes.
Operation method
basic program
Materials and Instruments
Organ Move 1. Place the dissected organ or embryo in a labeled 20 ml screw-top glass vial (silanized glass vials are used exclusively for small sample quantities), add 4% PFA freshly prepared at 4°C, and fix at 4°C for the desired time.2. Melt the paraffin in an oven at 60°C. Caveat 1. Xylene is a toxic organic solvent, steps 6 and 7 should be carried out in a fume hood. 2. The ninth step should be operated quickly to prevent the paraffin from condensing, if condensation occurs, the paraffin should be re-melted at 60 ℃ before the start of the 1 h incubation. For more product details, please visit Aladdin Scientific website.
Paraformaldehyde Paraffin Wax Transferred Xylene
Glass bottles Incubators Pasteur pipettes Embedding rings Embedding molds
3. After fixation, pour off the fixative (taking care not to lose the sample) and replace it with 50% ethanol. Immediately replace with fresh ethanol and dehydrate 3 times in 50% ethanol for 20 min each time at room temperature, followed by 3 times in 70% ethanol for 20 min each time at room temperature (samples can be stored in 70% ethanol at 4°C for several days).
4. Place the sample in 95% ethanol twice for 20 min each time at room temperature.
5. Place the sample in 100% ethanol twice for 20 min each time at room temperature.6. Exchange 100% ethanol with xylene, then immediately replace with fresh xylene and leave in xylene 2 times for 10 min each.7. Pour off xylene and add 5 fresh xylene to each sample vial, add an equal amount of melted paraffin wax using a hot glass pipette (work quickly to prevent condensation), mix well and leave the samples at room temperature overnight.
8. Melt the paraffin/xylene mixture by placing the sample bottle in a 60°C oven. Prepare a 60°C heating block with holes for 20 ml sample bottles and when the paraffin/xylene mixture has melted, transfer the bottles to the heating block.
9. Pour the paraffin/xylene mixture into the waste bottle and immediately add the newly melted paraffin with a hot glass pipette. Return the sample bottle to the heating block. Repeat this step, one by one, for each sample. Place the sample bottle back into the 60°C incubator and incubate for 1 h.10. Remove the sample vials from the warming chamber and place them back in the 60°C heating block, repeating step 9 twice for a total incubation time of 3 h.11. Prepare the embedding mold (e.g., for silanization spraying) according to the manufacturer's instructions, add melted paraffin to the mold with a hot glass pipette, and immediately transfer the sample to the paraffin-filled mold with a hot tweezer or a capillary pipette with the end cut off.12. Place an embedding ring on the mold with paraffin wax and write a label on the ring for future sample identification. A hot, stretched and sealed pipette may be used to adjust the position of the sample in the mold, and the module may be allowed to set at room temperature to allow for complete condensation.
13. Remove the wax block from the embedding mold, place it in a dry place at room temperature and slice.