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Essential Tips | Why Do Others Achieve Such Beautiful Stained Sections? Sharing Techniques for Histological Staining

Slice staining, as the most commonly performed pathological analysis, is undoubtedly something that many of you have tried. However, the results can vary widely: while some achieve exceptionally clear and well‑defined outcomes, others find themselves consistently struggling to get satisfactory results.

Today, we’re sharing some handy tips for section staining.

 

Paraffin section

1. Material Selection

(1) Specimen collection must be rapid; prolonged exposure can lead to alterations in the cellular composition and structure of the tissue.

(2) During tissue sampling, minimize damage to the sampled tissue block as much as possible.

2. Fixed

(1) Fixatives should be prepared fresh as needed; some mixed fixatives may undergo redox reactions, and premature preparation can compromise their fixing efficacy. After preparation, store the solution in a cool, shaded location, away from direct sunlight.

(2) The fixative must be used in sufficient quantity—typically 20 to 30 times the volume of the tissue block—and for tissues with high water content, the fixative should be replaced 1–2 times during the process.

(3) After securing the sample, store it in a tightly sealed or capped container and clearly label both the inside and outside of the container.

(4) Different tissues require different fixatives; for example, the eyeball has a specialized fixative. The choice of fixative can influence subsequent sectioning and staining.

3. Dehydration

(1) Dehydration must be carried out in a covered glass bottle.

(2) When replacing the high-concentration dehydrating agent, it is best not to move the tissue block.

(3) The residence time at each stage should not be excessively long, and the dewatering duration must be strictly controlled.

(4) If the dehydrated tissue blocks need to be stored overnight, they may be kept in a 70% ethanol solution.

(5) Dehydration must be thorough; otherwise, transparency will be difficult to achieve.

4. Transparent

(1) When using a clearing agent, always keep the container tightly sealed to prevent moisture from the air from entering.

(2) The replacement of the clearing agent at each stage must be performed quickly to prevent moisture absorption.

(3) During the clearing process, if a white haze appears around the material, it indicates that the water in the material has not been completely removed; the material should be re‑dehydrated before proceeding with clearing.

5. Wax Penetration

(1) Conduct the process at as low a temperature as possible, ensuring that the paraffin does not solidify.

(2) The wax‑penetrating temperature must be kept constant; it should not fluctuate.

(3) Operations should be performed swiftly, completing the paraffin infiltration process in the shortest possible time to prevent tissue hardening, embrittlement, and shrinkage.

6. Slicing

(1) Before each use of the microtome blade and specimen, or when changing specimens, be sure to lock the handwheel and cover the blade with the blade guard.

(2) Position the knife holder on the base as close to the specimen as possible, and adjust the specimen’s surface orientation so that it is as parallel to the blade as feasible.

(3) During sectioning, turn the handwheel at a constant speed. When cutting through relatively hard specimens, reduce the speed of the handwheel.

(4) Before operating the microtome and specimen, or when changing specimens or the microtome blade, or during breaks, the handwheel must be locked, and the blade edge must be covered with the blade guard.

 

Frozen section

1. The amount of embedding agent must be appropriate; too much or too little will both compromise the quality of tissue cryopreservation.

2. First, dispense a small amount of glue onto the holder and pre‑freeze it for approximately 30 seconds, allowing the glue to solidify before placing the tissue. When positioning the tissue, apply additional glue around its perimeter to ensure proper freezing.

3. The freezing temperature must be adjusted according to the specific tissue type; both excessively low and excessively high temperatures can adversely affect subsequent sectioning of the tissue block.

4. The slides may be stored at room temperature; refrigeration is not required.

5. Keep your blade sharp when slicing. If you notice a decline in slice quality, we recommend replacing the blade. Additionally, adopt the most comfortable and relaxed posture to help maintain better control of both hands.

6. You can use a brush to help remove the slices.

7. When slicing, continuously and evenly rotate the slicer; this produces a better result than stopping after each slice.

 

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Dyeing methods are diverse, and machine dyeing generally yields better results. Below is a summary of several common dyeing procedures.

HE Staining Procedure

1. Deparaffinize the sections in xylene I for 5 min.

2. Xylene II dewaxing, 5 min

3. Xylene III dewaxing, 5 min

4. Anhydrous ethanol I 1 min

5. Anhydrous ethanol II, 1 min

6. 95% ethanol for 1 min

7. 85% ethanol for 1 min

8. Rinse with tap water for 1 minute

9. Hematoxylin stain 5–10 min

10. Rinse with tap water, moderately

11. Differentiate in 1% hydrochloric acid–alcohol for a few seconds.

12. Rinse with tap water, moderately

13. Eosin stain, 1–3 min

14. Rinse with tap water, moderately

15. 95% ethanol I 10s

16. 95% Ethanol II 10s

17. Anhydrous ethanol I 30s

18. Anhydrous Ethanol II 30s

19. Anhydrous ethanol III, 1 min

20. Xylene Clear I 1 min

21. Xylene Clear II 1 min

22. Xylene Clear III 1 min

23. Resin Mounting

 

Sirius Red staining

1. Dewax paraffin sections to water

2. Sirius Red stain for 1 hour

3. Rinse under running water for several seconds.

4. Differentiate in 0.5% acetic acid for 30 seconds

5. Anhydrous ethanol, 30 s × 3

6. Xylene 1 min × 3

 

PAS staining

1. Dewax paraffin sections to water

2. Oxidize with periodic acid for 20 min; rinse with distilled water for 3 min.

3. Schiff’s reagent for 40 min; distilled water for 1 min

4. Hematoxylin for 5 min; distilled water for 2 min

5. Hydrochloric acid–alcohol for 1 second; distilled water for 2 minutes

6. Blueing solution 5 s

7. Anhydrous ethanol, 30 s

8. Xylene for 3 min

 

Masson's trichrome stain

1. Dewax paraffin sections to water

2. Hematoxylin for 5 min; hydrochloric acid–alcohol for 10 s

3. Blueing solution for 10 s; distilled water for 2 min

4. Ponceau S for 5 min; 0.2% glacial acetic acid for 1 min

5. 1% phosphomolybdic acid for 1 min; aniline blue for 1 min

6. 0.2% acetic acid for 1 min

7. Anhydrous ethanol, 30 s

8. Xylene for 3 min

 

Effect Demonstration

H&E staining results of mouse tissues

CD71 single-label immunofluorescence of tumor tissue

 

Aniphe BioLab can provide you with comprehensive experimental services, including animal model development and pathological analysis.

A professional team and cutting-edge expertise deliver quality assurance.

A variety of staining methods, immunohistochemistry, and pathological imaging services are available at affordable prices. Feel free to inquire!

 

 

Organized by | Aniphe BioLab Marketing Department

Supervisor  | Always

Editor-in-Chief | Lemon

Editor-in-Charge | Little Swallow

Editor | Lemon

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