Tissue Fixation Protocol

Tissue preparation and fixation are foundational to histology, ensuring the preservation of tissue architecture and cellular integrity crucial for generating high-quality slides. These steps prevent autolysis and degradation, which can otherwise compromise the tissue’s morphological features and obscure critical biological insights. Proper tissue preparation and fixation not only prevents decay but also enhances the tissue’s ability to withstand subsequent processing steps, such as sectioning and staining. If your project involves, precision histology, antibody optimization, IHC, IF, or RNA in situ hybridization (F/ISH), the preparation steps ultimately contributes to the reliability and reproducibility of the readouts such as the pathology review or the data generated through image analysis.

Tissue Preparation and Fixation for FFPE Processing

• Remove tissue from host and wash in phosphate-buffered saline (PBS).
• Tissue samples should be placed in fixative as soon as possible. Neutral Buffered 10% Formalin (10% NBF) at room temperature or fresh 4% paraformaldehyde (4% PFA) at 4°C could be used for most routine paraffin processing at a ratio of 20:1, i.e. 20 mL to 1 cm3 of tissue at necropsy. Some tissue types such as brains, eyes, lungs, intestines, bones and rodent embryos may require different fixatives and/or special processing (refer to Table 1).
• Large tissues should be trimmed no larger than 3-5 mm thick immediately after necropsy for proper penetration of fixative and placed in fixative for 24-48 hours at room temperature.
• Once tissue is fixed, transfer to 70% EtOH prior to shipment. Cover sample containers with parafilm prior to shipment to GlintLab to prevent leakage. If tissue is being shipped in fixative, place samples in parafilmed containers in Styrofoam or secondary containment for shipping.
• Tissue Trimming will be performed by GlintLab upon receipt of samples (as needed).
  • Standard trimming of rodent tissues will be performed by our histologists based on RITA and NACAD guidelines for organ sampling and trimming in rats and mice.
  • Custom trimming can also be performed by our histologists. Please contact your Study Coordinator  if you have special trimming instructions.

Tissue	Special Processing	Fixative
Brains	N/A	4% PFA at 4°C, transfer to PBS, ship at 4°C
Eyes	N/A	Davidson’s
Lungs	Inflate lungs (refer to section B) before fixation	10% NBF
GI/Intestines	Rinse well with PBS, rolled or unrolled, before fixation	10% NBF
Bones	Fix bones, then decalcify (refer to section C)	10% NBF

Lung Inflation and Fixation for FFPE Processing

• Lung inflation is performed for best morphology. Perfuse the lungs gently through the trachea with fixative until the lungs are fully expanded to a normal level as expected to fill the chest cavity.
• Place sample in fixative for 24-48 hours at room temperature.
• Once tissue is fixed, transfer to 70% EtOH prior to shipment. Cover sample containers with parafilm prior to shipment to Glint.

Bone Decalcification (Glint Lab offers these services)

• Bone decalcification must be performed to remove minerals from fixed bone before it is malleable for trimming, processing, and sectioning.
• Choose the appropriate decalcification method for your sample.
  • We recommend using the Immunocal™. Decalcification with Immunocal™ Decalcifier helps preserve both tissue morphology and antigenicity, ensuring optimal results for immunohistochemistry (IHC) staining.
  • Alternative solution:
    • HCl/Formic Acid: Fast decalcification and samples can be used for most special stains such as H&E staining, not recommended for IHC staining.
    • 14% EDTA, tetra-sodium, pH 7.3-7.6 made with HCl or acetic acid: Slower decalcification than acid decalcifying agents. Samples can be used for IHC staining.
• Remove all skin, fur and muscle tissues, place the bone region of interest into selected decalcifier at a ratio of 20:1, i.e. 20 mL to 1 cm3 of tissue with occasional swirling of the jar or gentle agitation.
• Length of decalcification is dependent on tissue size, and most mouse tissue will decalcify in 2-3 days with HCl/Formic Acid. When is bone sample is malleable, the decalcification process is complete.
• Please contact your Study Coordinator  if you have never decalcified tissue before and/or would like us to perform the bone decalcification.

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