Paraffin section preparation steps, one, reagent matching method
1. Neutral formaldehyde fixative:
Formaldehyde (37%-40%, commercially available, we will buy this concentration) 100mL
Disodium hydrogen phosphate 6.5
Potassium dihydrogen phosphate (sodium) 4g
Double distilled water 900mL
2. Hematoxylin and Eosin staining solution are Biyuntian products
3.1% hydrochloric acid ethanol solution
1 part of hydrochloric acid
70% alcohol 100 parts
4. Glycerin tablets:
Protein 50 ml
Glycerin 50 ml
Sodium salicylate (preservative) 1g
When preparing, break one egg into a bowl or cup, leave the egg white to leave the egg white, use a glass rod to make a snowflake foam, then filter it into a measuring cylinder with coarse paper or double gauze. After several hours or overnight, you can filter it. Clear protein solution. At this time, an equal amount of glycerin was added thereto, and the mixture was slightly shaken to mix the two. Finally, a preservative (sodium salicylate) is added for antiseptic. Can be kept for a few months.
Paraffin sectioning steps,
Second, the experimental steps1. Material
The mice were sacrificed by cervical dislocation, the abdominal cavity was opened, and liver tissue (or other tissues) was cut.
The cut tissue block should not be too large to facilitate the penetration of the fixative, and is usually 5 mm × 5 mm × 2 mm or 10 mm × 10 mm × 2 mm. Remove the required liver tissue and cut into small pieces 2-3 mm thick.
Precautions:
(1) The action of taking materials should be rapid, and it is not appropriate to delay for too long to avoid changes in the composition and structure of the tissue cells.
(2) The sectioning material should be selected according to the part to be observed, so as not to damage the required part as much as possible.
2. fixed
The cut liver tissue was washed with physiological saline tissue, and immediately placed in a neutral formalin fixative for 30-50 min.
Precautions:
(1) Generally, the fixed liquids are all better with new ones. After being prepared, they should be stored in a cool place. They should not be placed in the sun to avoid chemical changes and lose their fixation.
(2) Oxidation-reduction occurs between the components of some mixed fixatives. It must be mixed before use. If the mixing is too early, it will have no effect when it is fixed.
(3) When fixing the material, the fixing liquid must be sufficient, generally 20~30 times of the material block. Some materials with more moisture should be replaced with 1-2 times of new liquid in the middle.
(4) After the material is fixed, store it in a tightly packed or capped container, and label the outside of the container, and put the corresponding label into the solution with the same material to avoid confusion. The label indicates the fixative, material source, date, etc. The text on the label, written in black pencil or drawing black ink.
3. Washing
After the material has been fixed, rinse with running water for several hours or overnight.
Dehydration
The material was dehydrated by 70%, 80%, 90% ethanol solution in each stage, each for 30 minutes, and then placed in 95% and 100% twice for 20 minutes each time. each
Paraffin sectioning steps,
Precautions:
(1) Dehydration must be carried out in a covered glass to prevent absorption of moisture from the air.
(2) When replacing the high-grade dehydrating agent, it is best not to move the material to avoid damage. Use a pipette to suck out the dehydrating agent in the vessel, then use the water to absorb the remaining liquid in the vessel, and then add a high-grade dehydrating agent to the vessel. .
(3) In low-concentration alcohol, the retention of each stage should not be too long, otherwise it will easily soften the tissue and promote the disintegration of the material.
(4) In high concentration or pure alcohol, the time of each stage should not be too long, otherwise the tissue will become brittle and affect the sectioning.
(5) If you need to stay overnight, stay in 70% alcohol.
(6) Dehydration must be thorough, otherwise it is not easy to be transparent, and even white turbidity appears in the transparent agent.
5. Transparent
Mixture of pure alcohol and xylene in 15 minutes, xylene I15min, II15min (to transparent).
Since ethanol is incompatible with paraffin, and xylene is soluble in both ethanol and paraffin, it undergoes a transition through xylene after dehydration. When all of the tissue is occupied by xylene, the light can pass through and the tissue exhibits varying degrees of transparency.
Paraffin sectioning steps,
Transparent considerations
(1) When using a clearing agent, always close the lid to prevent moisture from entering the air.
(2) Replace each level of transparent agent, the action should be rapid, on the one hand, in order not to dry the material block, on the other hand, to avoid moisture absorption.
(3) In the process of transparency, if there is a white mist around the material, it means that the water in the material is not removed, it should be returned to pure alcohol and dehydrated again, then transparent.
6. Wax penetration
A mixture of xylene and paraffin was placed for 15 min, and then paraffin I and paraffin II were added for 50-60 minutes each.
The purpose of the wax penetration is to remove the clearing agent (such as xylene, etc.) in the tissue, so that the paraffin penetrates into the interior of the tissue to a degree of saturation for embedding. The wax penetration time depends on the size of the tissue. The wax should be carried out in the incubator, and keep the temperature inside the box at 55-60 °C, pay attention to the temperature is not too high, so as to avoid brittle tissue. Usually placed in the incubator for 0.5h.
Paraffin sectioning steps,
Wax precautions
(1) Try to keep it at a lower temperature, so that the paraffin does not solidify;
(2) The temperature of the wax should be constant and should not be high or low;
(3) The operation should be rapid, and strive to complete the paraffin penetration process in the shortest time to avoid causing the tissue to become hard, brittle, and shrink.
7. Embedding
When embedding, use a tweezers to pick up the paraffin mold (metal texture) and heat it slightly on the alcohol lamp. Place it on a flat tabletop, remove the wax cup containing pure paraffin from the incubator, and pour a little paraffin. Then heat the tweezers slightly on the alcohol lamp, and take the material into the wax mold with the cut surface facing down, neatly arranged. Place the cassette and gently pour the melted wax.
8. Slice
(1) Mount the fixed and repaired paraffin block on the table of the microtome.
(2) Fix the slicing knife on the holder and the knife edge is upward.
(3) Shake the screw to make the paraffin block close to the knife edge, but not beyond the knife edge.
(4) Adjust the angle and position between the paraffin block and the knife edge, and the blade and the paraffin slice are about 15 degrees.
(5) Adjust the thickness adjuster to the desired slice thickness, typically 4-10 microns.
(6) After everything is adjusted, the Lord can start slicing. At this time, shake the runner in the right hand, cut the wax block into a wax belt, and lift the wax belt with the brush on the left. The rocking speed should not be too fast, usually 40-50r/min.
(7) When the cut wax strip is 20-30cm long, the right hand gently picks up the wax strip with another brush to avoid curling and pulling into a belt, which is placed flat on the wax belt box, on the side of the knife side. Smoother, face down, wrinkled side up.
(8) Cut a small piece of wax with a single-sided blade, add a drop of water to the glass, and observe whether the slice is good under a magnifying glass or microscope.
(9) After the slicing work is finished, the slicing knife should be removed and the paraffin wax on the knife should be wiped off, and the microtome should be wiped clean and stored.
9. Exhibitions, patches
Open the water bath to maintain the water temperature at 40-45 ° C, and prepare a 30% ethanol solution.
(1) When slicing, place a bowl of 30% ethanol solution on the table next to the microtome.
(2) Using a small forceps, take the wax tape cut in advance with a blade and place it on the water surface of the ethanol solution to spread the slice.
(3) The scorpion gently separates the pieces that are joined together, and the slice is completed with a slide, and the unfolded slice is taken to warm water to fully spread.
(4) Take another clean glass slide and pick up the unfolded slice so that it is located at 1/3 of the slice. The other end (edge, rough end) is marked or labeled on the polished surface and placed on the slicer. .
10. Dewaxing and rehydrating
The temperature of the water bath was adjusted to 60 ° C. When the water temperature was controlled at 60 ° C, the slice was placed in a dry dyeing tank together with the slice holder, placed in a water bath, and covered with a lid (sealable), and the wax was melted for 30 minutes.
After that, the paraffin sections were dewaxed by xylene I and II for 5 min, then placed in 100%, 95%, 90%, 80%, 70% alcoholic solutions for 3-5 min, and then placed in distilled water for 3 min.
11. dyeing
The sections were stained in hematoxylin for about 10-30 min.
The dyeing time should be shortened or extended as appropriate according to the maturity of the dye and the room temperature. When the room temperature is high, the dyeing is promoted, and the dyeing time can be shorter. Otherwise, the time can be extended appropriately. When the room temperature is low in winter, it can be dyed in an incubator.
12. Washed
Rinse with running water for about 15 minutes. Make the color of the slice blue (or put it in alkaline water), but be careful not to over-flow the water to prevent the slice from falling off.
13. Differentiation
The sections were immersed in 1% hydrochloric acid in ethanol for about 2 seconds to several tens of seconds. See that the slice turns red and the color is lighter.
14. rinsing
Slice and place in tap water to make it blue.
15. Dehydration I
Slice into 50% ethanol → 70% ethanol → 80% ethanol for 3-5 min.
16, counterstaining
The staining was performed for 1-3 min with 0.5% eosin ethanol solution.
Eosin mainly stains the cytoplasm, and the coloration should be matched with the density of the hematoxylin-stained nuclei. If the nuclei are thicker, the cytoplasm should be densely stained to obtain a sharp contrast. Conversely, if the nucleus is stained lightly, the cytoplasm should also be lightly stained. A few drops of glacial acetic acid can be added to the eosin ethanol solution to promote the cytoplasm to be easily colored, and it is not easy to fade when dehydrated by ethanol.
17. Dehydration II
The sections were placed in 95% ethanol and the excess red was washed off and then placed in absolute ethanol for 3-5 min. Finally, blot excess ethanol with absorbent paper.
18. Transparent
The sections were placed in xylenes I and II for 3-5 min each.
The xylene should be kept as dry as possible, and should be replaced frequently, or wrapped in gauze with anhydrous copper sulfate into the dyeing tank to absorb moisture. Slices such as white fog appear in xylene, indicating that dehydration is not completed, should be returned to ethanol to re-dehydrate, otherwise the slice is difficult to microscopic examination.
19. Sealing: Neutral gum seal
Since the section is transparent to xylene, a neutral gum is used as a sealing agent, and the gum can be diluted with xylene to a suitable consistency.
Method of sealing:
Cover sheets of different specifications should be selected according to the size of the material before sealing. After the material is transparent, put a clean absorbent paper on the table, remove the slide containing the material from the xylene and put it on the paper (the side of the slice is up), and quickly drop a drop of gum in the center of the slice. Do not allow xylene to dry before proceeding. Gently grip the right side of the coverslip with a small hand-held tweezers, slightly tilt it to the left side with the sealing agent, and then slowly lower the coverslip. This will reduce or avoid the generation of air bubbles. If the glue is not enough, you can use a glass stick to add another drop of gum from the edge of the coverslip. If there is too much glue, it can be scraped off with a knife after drying, and the residual gum is wiped off with gauze and xylene.
Staining results: the nucleus was stained blue by hematoxylin and the cytoplasm was stained with eosin to pink.
Paraffin sectioning steps
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