Common Issues in Immunohistochemical Tissue Processing

1. 10% Neutral Buffered Formalin Fixative: 10mL of 40% formaldehyde + 90mL of 0.01M pH7.4 PBS. This is the universally recommended fixative for immunohistochemistry abroad.

2. 4% Paraformaldehyde Phosphate Buffer Solution: Mix 40g of paraformaldehyde with 500mL of 0.1M pH7.4 PB solution, heat to 60°C, stir and add 1N NaOH dropwise until the solution becomes clear. After cooling, add PB solution to a total volume of 1000mL and mix thoroughly.

3. B5 (Sodium Acetate-Mercuric Chloride-Formaldehyde) Fixative: 1.25g of anhydrous sodium acetate + 6.0g of mercuric chloride + 90mL of distilled water. Mix and dissolve these three components, then add 10mL of 40% formaldehyde before use. This solution is often used for fixing lymphoid tissues, and mercury precipitation removal should be performed before staining.

4. 95% Ethanol.

1. For surgical or biopsy specimens, strive to keep the tissue fresh, avoid drying, and fix as soon as possible.

2. The tissue block size should be approximately 2cm × 1.5cm × 0.2-0.3cm, with thickness especially controlled within 0.3cm.

3. The volume of fixative must be sufficient, generally more than 20 times the volume of the tissue.

4. Fixation time: 4-6 hours for small specimens, 18-24 hours or longer for large specimens. However, over-fixation can reduce antigen activity.

5. After fixation, tissues should be thoroughly washed with water to reduce artifacts caused by the fixative.

III. Requirements for Paraffin Sections　　

The advantages of paraffin sections are good preservation of tissue structure, ability for serial sectioning, clear tissue architecture, accurate antigen localization, and significant practical value in pathological diagnosis and retrospective studies. The preparation of paraffin sections for immunohistochemistry differs slightly from routine HE slide preparation:

1. During the infiltration and embedding process, paraffin should be kept below 60°C as much as possible.

2. Section thickness: 3-5μm; sections of lymphoid and renal biopsy tissues can be thinner. The cut paraffin sections should be intact, free from knife marks, chatter marks, folds, cracks, or loosening.

3. Section flattening can be performed using the double-floating method.

4. Slides must undergo anti-detachment treatment (Mai Xin product number: SLI-2001).

Note: The double-floating method refers to floating the tissue section in a 30%-40% ethanol solution for the first flattening during the flattening process, then retrieving the section and placing it in a water bath at 45°C-50°C for the second flattening. The advantage of this method is the tension difference between the ethanol solution and water, resulting in flat, wrinkle-free tissue sections. However, tissues with low intercellular adhesion, such as fat, may disperse upon contact with ethanol, so this method is not recommended for them. The concentration of ethanol and the temperature of the water bath can be adjusted accordingly based on the tissue type and paraffin melting point.

1. Cleaning of slides: Soak slides in potassium dichromate cleaning solution for 24 hours, rinse thoroughly with tap water, then wash with distilled water at least twice. Subsequently, soak the slides in 95% ethanol solution for 2 hours, remove, and dry in an oven.

2. Coating slides with anti-detachment adhesive: Common adhesives are poly-L-lysine or APES. For specific operating steps, refer to “Instructions for Use of Anti-Detachment Agents.”
3. Checking if slides are clean: Dip a cleaned slide into the ready-to-use adhesive solution. If the slide is clean, the adhesive will form a uniform, thin film covering the slide surface upon removal. If it flows off like running water, it indicates the slide is not clean and needs re-cleaning.

To effectively store immunohistochemistry reagents and maximize their shelf life, please note the following:

1. Upon receiving the reagents, check against the delivery list. If there are any discrepancies, please contact our company within 3 days.

2. After verification, store the reagents in a 4°C refrigerator as soon as possible.

3. For those who purchase concentrated reagents: Due to the small packaging size, briefly centrifuge before opening the cap to settle the reagent at the bottom of the vial, preventing waste from reagent sticking to the cap.

4. During experimental procedures, promptly return any remaining reagents to their original storage location after use. Reagents left at room temperature for too long may lose potency, and chromogenic reagents exposed to light for extended periods are also prone to degradation.