Preparation of paraffin sections (hematoxylin-eosin counterstaining method)
Preparation of paraffin sections (hematoxylin-eosin counterstaining method)
Paraffin section making can be used to (1) maintain the normal interrelationships between cells, and can retain the original appearance of cells better and for a longer period of time. (2) It is the main method of preparation for light microscopy.
Operation method
Preparation of paraffin sections (hematoxylin-eosin counterstaining method)
Principle
Paraffin sectioning is the most basic sectioning technique, and frozen sections and ultrathin sections, etc. have been developed on the basis of paraffin sections. Hematoxylin and eosin contrast staining (H.E. contrast staining for short) is the most commonly used staining method for tissue sections. This method has a wide range of application, and it can color various components of tissue cells, which is convenient for comprehensive observation of tissue structure, and it is also suitable for various materials fixed with fixative, and it is not easy to fade and can be preserved for a long time after staining. After HE staining, the nucleus is stained blue-purple by hematoxylin and the cytoplasm is stained pink by eosin.
Materials and Instruments
Rat Liver Kidney Myocardium Skeletal muscle or other tissues; soybean or wheat Green bean Onion Garlic Roots, stems, and leaves of broad beans Move A reagent preparation method Caveat 1. After hematoxylin staining, color separation is crucial and should be done under the microscope. Generally, it is appropriate that the nucleus staining is relatively clear and the cytoplasm and other basic colorless. If overstaining is found, the color separation time can be extended. If the staining is too shallow, it should be re-stained before color separation. 2、If the staining of Eosin is found to be too deep, the color separation time in 95% alcohol can be extended. 3、After the section is dehydrated by alcohol, if there is turbidity or white opacity when transferring to xylene, it means that the dehydration is not complete, and the section should be returned to anhydrous alcohol immediately for rehydration, and the anhydrous alcohol should be replaced if turbidity still occurs. 4、The time required for each step of staining is often affected by the temperature and thickness of the section, which can be adjusted according to the observation under the mirror. 5、If the staining reaction is not normal, check whether the dye solution and reagents are invalid or misused. Common Problems Possible problems and solutions for paraffin slices Disadvantage 1: Paraffin tape is not straight Cause: 1. The top and bottom sides of the paraffin block are not parallel to each other. 2. The top and bottom sides of the paraffin block are not parallel to the cutting edge. 3. The sharpness of the cutter is not uniform, resulting in localized differences. 4. One side of the block is softer than the other, or the hardness of the two sides is not the same. 5. The material is not centered in the block. 6. The material is too large and misshapen. Remedy 1. Remove the wood from the table and dry both sides. 2. Adjust the specimen table so that they are parallel. 3. Move the blade and use a new one. 4. Allow the wax block to cool before cutting, or re-embedded 5. cut away part of the paraffin with the blade to center the material 6. Cut off a little paraffin on the larger side. Disadvantage #2: Slices separate and do not join into bands Reasons: 1. Room temperature is too low 2. Paraffin wax is too hard 3. Too little wax left on the edge of the material 4. The angle of the knife is not suitable Remedy : 1. Turn on an electric light above the slicer or raise the room temperature. 2. Add a layer of 45℃ soft wax on the surface of the wax block or rub the surface of the block with finger wax. 3. Re-embedding 4. Correct the angle of the knife Disadvantage 3: Sections rolled up in a cylindrical shape Causes: 1. Room temperature is too low 2. Paraffin too hard 3. The knife is too blunt 4. Knife tilt angle is too large Remedy : 1. Increase the room temperature 2. Add soft wax 3. Use a brush to spread the wax sheet and press it down, cut 2-3 slices to form a strip. 4. Reduce the angle of inclination Disadvantage 4: The slices stick to the slicing knife and crumple together. Reason: 1. Room temperature too high 2. Paraffin wax is too soft 3. A layer of paraffin is left on the knife edge 4. Dull cutting edge Remedy : 1. Lower the room temperature 2. Put the wax block into the cool water slightly immersed Lei-increased 3. Slice the thickness, change to hard wax embedding, use xylene to wipe off the paraffin on the knife edge. 4. Sharpen or move the knife Disadvantage 5: Longitudinal cracking of slices Causes: 1. Knife is notched 2. Paraffin block contains particles, impurities 3. The knife left debris or filaments 4. Tissue is too hard Remedy : 1. Move the knife 2. Remove the particles 3. Clean the incision 4. Let the embedded block soak in water Disadvantage 6: Sections have horizontal lines Causes: 1. Fixing screws of knife and table wood are too loose 2. The tilt of the knife is too big 3. Paraffin chips on the knife edge. Remedy: 1. Tighten the screw 2. Flatten the knife 1-2 times before cutting. 3. Wipe off with chloroform Disadvantage 7: uneven thickness of slices Reason: 1. Slicer defective 2. Improperly clamped knife 3. Failure to tighten the specimen table spiral 4. Paraffin block is too hard Remedy : 1. Correct the slicer device 2. Symptomatic treatment 3. Tighten the screw 4. Soak the paraffin blocks in water. Disadvantage 8: Each slice is not uniformly thick or thin Causes: 1. Knife vibration is due to the material being too hard 2. The inclination of the knife is too large Remedy : 1. Apply a layer of fire-wool glue on the surface of the wax block 2. Reduce the inclination angle Disadvantage 9: The material is cracked and broken or peeled off Cause: 1. Unclean dehydration 2. There is transparent agent residue 3. Paraffin penetrates when the temperature is too high or time is too long 4. Due to the influence of dehydrating agent and transparent agent, the tissue becomes hard and brittle 5. The material is too hard or too coarse Remedy: 1. Cannot be remedied 2. Increase the time of wax dipping and re-embedding. 3. Cannot be remedied 4. Dehydrate and make transparent with n-butanol, tert-butanol and dioxane, etc. 5. Apply a thin layer of fire wool gel solution on the surface of the wax block Disadvantage 10: Rustling occurs during sectioning Causes: 1. Tissue block is too hard 2. Embedding temperature is too high 3. Crystals remain in the material Remedy : 1. Soak in water to soften 2. Cannot be remedied 3. Unable to remedy Disadvantage XI : Paraffin blocks lift the wax tape up Cause: 1. Due to static charge due to friction 2. Paraffin fragments are attached to the paraffin block 3. Paraffin debris on the knife edge Remedy : 1. Increase the room temperature 2. Remove the debris with a razor blade. 3. Remove with xylene or oxoform For more product details, please visit Aladdin Scientific website.
Neutral formalin fixative, various concentrations of alcohol, xylene, paraffin, beeswax, hematoxylin stain 1% eosin alcoholic solution 1% hydrochloric acid alcoholic solution Glycerol protein mucoadhesive Neutral gum. Carnot fixing solution Ehrlich hematoxylin dyeing solution 1% eosin alcohol solution 1% hydrochloric acid alcohol solution Various grades of alcohol (30%, 50%, 70%, 80%, 90%, 95%, 100%) Xylene Glycerine protein mucoadhesive tablets
Slicing knife Slicer Thermostat Wax cup Alcohol lamp Dissecting knife Dissecting scissors Dissecting tray Petri dish Tweezers Single sided blade Brush Embedding box Staining vat Coverslip Slide Slide mounted slide Tray Resin Gum Resin bottle Microscope Thermometer Basin Water bath Warming table Scissors Dissecting needle Small table wood Staining vat Beaker Basin Molten wax oven
1. Neutral formaldehyde fixative:
formaldehyde (37%-40%, commercially available, the Institute to buy that concentration) 100mL
disodium phosphate 6.5
potassium dihydrogen phosphate (sodium) 4g
double-distilled water 900mL
2. Hematoxylin, eosin staining solution for the products of BiyunTian
3. 1% hydrochloric acid alcohol
hydrochloric acid 1
70% alcohol 100
4. Glycerol protein patches:
egg white 50 ml
glycerol 50 ml sodium salicylate (preservative) 1g preparation, a broken egg yolk and leave the egg white to snow-like foam, then mixed with a glass rod to beat 50 ml
sodium salicylate (preservative) 1g
Preparation of an egg broken into a bowl or cup, yolks to leave the egg white, using a glass rod to beat the snowflake foam, and then filtered through coarse paper or double gauze to the measuring cylinder, after a few hours or overnight, can be filtered out of the transparent egg white liquid. At this point, add an equal amount of glycerin to it and shake slightly to mix the two. Finally, a preservative (sodium salicylate) is added for preservation. Can be stored for several months.
Experimental steps
1. Sampling
Cervical dislocation method of execution of mice, open the abdominal cavity, cut the liver tissue (or other tissues).
The cut tissue block should not be too large for the fixative to penetrate, usually 5 mm × 5 mm × 2 mm or 10 mm × 10 mm × 2 mm is appropriate. Remove the desired liver tissue and cut it into a small piece 2-3 mm thick.
Precautions:
(1) The action of removing the material should be rapid, and should not be made too long a delay to avoid changes in the composition and structure of the tissue cells.
(2) The slicing material should be selected according to the part to be observed, and the needed part should not be damaged as much as possible.
2. Fixation
The cut liver tissue should be washed with saline, and then put into neutral formalin fixative to fix it for 30-50min.
Note:
(1) Generally speaking, the fixative should be freshly prepared, and should be stored in the shade after preparation, and should not be put under sunlight to avoid causing damage. Should not be placed in the sun, so as not to cause chemical changes, the loss of fixed effect.
(2) Some of the components of the mixed fixative will occur between the redox effect, must be mixed before use, if the mix is too early, fixed when there is no effect.
(3) Fixed material, the fixing solution must be sufficient, generally for the material block of 20 ~ 30 times, some of the moisture of the material, the middle of the new liquid should be replaced 1-2 times.
(4) After the material is fixed, saved in a tight plug or cover the container, at the same time in the container outside on the label, and with the material in the solution into the corresponding label, so as to avoid mutual confusion. The label indicates the fixing solution, the source of the material, the date and so on. The text on the label should be written in black pencil or drawing black ink.
3. Washing
After the material is fixed, rinse it under running water for several hours or overnight.
4. Dewatering
The material should be dehydrated by 70%, 80%, 90% ethanol solution at all levels in sequence for 30min each, and then put into 95% and 100% twice each for 20min each time.
Precautions:
(1) Dewatering must be carried out in a covered glassware to prevent the absorption of moisture in the air. absorption of moisture in the air.
(2) When replacing the dehydrating agent of higher level, it is better not to move the material to avoid damage, use a pipette to suck out the dehydrating agent in the vessel, then use suction to suck out the remaining liquid in the vessel, and then add the dehydrating agent of higher level in the vessel.
(3) In low concentration of alcohol, each level should not stay too long, otherwise it is easy to soften the tissue and promote the disintegration of the material.
(4) In high concentration or pure alcohol, the stay at each level should also not be too long, otherwise it will make the tissue brittle and affect the sectioning.
(5) If an overnight stay is required, it should be in 70% alcohol.
(6) Dehydration must be thorough, otherwise it is not easy to be transparent, and even make white turbidity inside the transparent agent
5. Transparent
Pure alcohol, xylene, equal amount of mixture for 15min, xylene Ⅰ 15min, Ⅱ 15min (until transparent).
As ethanol and paraffin are not soluble, and xylene is soluble in both ethanol and paraffin, so after dehydration and also after xylene to transition. When the tissue is fully occupied by xylene, light can pass through and the tissue shows varying degrees of transparency.
Transparency Precautions
(1) When using the transparency agent, keep the lid tightly closed at all times to prevent moisture from entering the air.
(2) Replace each level of transparency agent, the action should be rapid, on the one hand, in order not to make the material block dry, on the other hand can avoid the absorption of moisture.
(3) In the process of transparency, if a white mist appears around the material, it means that the water in the material has not been removed, and it should be returned to pure alcohol for rehydration and then transparency.
6. Transparencies
Put in a mixture of half of xylene and half of paraffin wax for 15min, and then put in paraffin Ⅰ, paraffin Ⅱ to transparencies for 50-60min each.
The purpose of permeable wax is to remove the transparent agent (such as xylene, etc.) in the tissue, so that the paraffin wax can penetrate into the tissue to reach the saturation level for embedding. The duration of permeabilization depends on the size of the tissue. Transmission of wax should be carried out in a constant temperature box, and keep the temperature in the box at about 55-60 ℃, pay attention to the temperature is not too high, so as to avoid tissue brittle. Generally placed in the constant temperature box 0.5h.
Notes on wax penetration
(1) Try to keep in the lower temperature, to the extent that the paraffin wax does not solidify;
(2) The temperature of wax penetration should be constant, and should not be fluctuating;
(3) The operation should be rapid, and strive to complete the process of paraffin wax penetration in the shortest possible time, so as not to cause the tissue to become hard, brittle, shrinkage, etc..
7. Embedding
Embedding, use the forceps to clip paraffin wax molds (metal texture) in the Alcohol lamp slightly heated, placed on a flat tabletop, remove the wax cup containing pure paraffin from the warming box and pour a little paraffin into it. Warm the forceps slightly on the alcohol lamp and place the material, cut side down, in the wax mold, arranging it neatly. Place the embedding box again and gently pour in the molten wax.
8. Slicing
(1) Load the secured and repaired paraffin block on the clamping table of the slicer.
(2) Attach the slicing knife to the knife clamp with the blade pointing upward.
(3) Rock the push screw so that the paraffin block is close to, but not over, the knife.
(4) Adjust the angle and position between the paraffin block and the knife edge, with the blade at about 15 degrees to the paraffin slice.
(5) Adjust the thickness adjuster to the desired slice thickness, generally 4-10 microns.
(6) After all adjustments, the main can start slicing. At this time, the right hand shakes the rotating wheel, let the wax block cut into wax tape, the left hand holds the brush to lift the wax tape, the shaking speed should not be too rapid, usually 40-50r/min.
(7) When the cut wax tape is 20-30cm long, the right hand with another brush gently picks up the wax tape, so as not to curl, and traction into a band, placed flat on the box of wax tape, by the knife surface of the side of the smoother side, facing down, the more wrinkled side facing up.
(8) Cut a small section of the wax slice with a one-sided blade, place it on a carrier glass with a drop of water, and place it under a magnifying glass or microscope to see if the section is good.
(9) After the slicing work is finished, the slicing knife should be removed and the paraffin stained on the knife should be wiped off with chloroform, and the slicer should be wiped clean and stored properly.
9. Expanding Slices, Pasting
Turn on the water bath to maintain the water temperature at 40-45°C, and prepare a 30% ethanol solution.
(1) Place a bowl of 30% ethanol solution on the table next to the slicer while slicing.
(2) Use small tweezers to pick up the wax band, which was pre-cut with a razor blade, and place it on the surface of the ethanol solution to unfold the section.
(3) The small forceps gently separated the connected sections and used one slide to complete the section. The unfolded section was fished into warm water to fully unfold it.
(4) Take another clean slide, fish up the unfolded section so that it is located in 1/3 of the section, and the other end (grinding edge, rough end) grind the surface to mark or label it, and put it on the section holder.
10. Dewaxing and rehydration
Adjust the temperature of the water bath to 60℃, and when the water temperature is controlled at 60℃, put the sections with the section holder into a dry staining cylinder, put it into the water bath, cover it with a lid (can be sealed) for 30 min until the wax melted.
After that, the paraffin sections were deparaffinized by xylene I and II for 5 min each, and then put into 100%, 95%, 90%, 80%, 70% alcohol solutions at all levels for 3-5 min each, and then into distilled water for 3 min.
11. Staining
Sections were put into hematoxylin for staining for about 10-30 min.
The time of staining should be shortened or lengthened appropriately according to the maturity of the stain and the high or low room temperature. When the room temperature is high to promote staining, the staining time can be shorter, otherwise, the time can be extended appropriately, and when the room temperature is low in winter, the staining can be put into a thermostat.
12. Rinsing
Rinse with tap water for about 15 minutes, so that the color of the section becomes blue (or put into the alkaline water can be), but be careful to ensure that the water should not be too large, to prevent the section from falling off.
13. Differentiation
Put the section into 1% hydrochloric acid in alcohol to discolour for about 2 seconds to dozens of seconds. See the sections become red, the color is lighter.
14. Rinsing
The sections are then put into tap water to restore the blue color.
15. Dehydration Ⅰ
The sections are put into 50% ethanol → 70% ethanol → 80% ethanol for 3-5min each.
16. Re-staining
Comparison staining with 0.5% eosin in ethanol solution for 1-3min.
The eosin is mainly staining the cytoplasm, and the intensity of the coloring should be matched with that of the nuclei of the cells stained with hematoxylin, if the nuclei are stained more intensely, the cytoplasm of the cells will be stained more intensely, the cytoplasm will be stained more intensely. If the nucleus is stained more intensely, the cytoplasm should also be stained intensely to obtain a sharp contrast. On the contrary, if the nucleus is stained lightly, the cytoplasm should also be stained lightly. A few drops of glacial acetic acid can be added to the ethanol solution of eosin to help the staining, so that the cytoplasm can be easily stained and the color is not easily faded by ethanol dehydration.
17. Dehydration Ⅱ
Wash the sections in 95% ethanol to remove the excess red color, and then put them into anhydrous ethanol for 3-5min. Finally, use water-absorbent paper to absorb the excess ethanol.
18. Transparency
The sections are put into xylene Ⅰ and Ⅱ for 3-5min.
The xylene should be kept as free of water as possible, and should be changed frequently, or used as an alternative to the xylene. , which should be changed frequently, or anhydrous copper sulfate wrapped in gauze should be placed in the dye vat to absorb water. Sections that show white mist in xylene indicate that dehydration has not been completed and should be returned to ethanol for rehydration, otherwise the sections will be difficult to microscopically examine.
19. Sealing: Neutral gum sealing
Since the sections are transparent through xylene, neutral gum is used as a sealing agent, and the gum can be diluted with xylene to the right consistency.
Methods of sealing:
Before sealing the sections, cover slips of different sizes should be used depending on the size of the material. After the material is transparent, put a piece of clean absorbent paper on the table, take out the slide containing the material from xylene and put it on the paper (with the side of the slice upward), quickly put a drop of gum in the center of the slice (never wait for the drying of the xylene to take place), gently hold the right side of the coverslip with small forceps held in the right hand, slightly tilt it so that its left side meets with the angle of the sealer, and then slowly put the coverslip down, which can reduce or avoid the air bubbles. This will reduce or avoid air bubbles. If there is not enough glue, use a glass rod to add another drop of gum from the edge of the coverslip to make up the difference. If there is too much gum, use a knife to scrape it off after drying, and use gauze dipped in xylene to wipe off the residual gum.
Staining results: the nucleus was stained blue by hematoxylin, and the cytoplasm was stained pink by eosin.
Source : Practical Laboratory Techniques in Neurobiology