Tissue Fixation

Disclaimer

The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.

Abstract

First and most important - the original tissue sample must be of good quality. Factors such as warm

ischemic time, the time delay between tissue excision and fixation, etc. are important. Ideally, tissues

should be acquired as close to still being viable as possible, and put into fixative as soon as possible

following excision. Delays lead to cell death, autolysis, and loss of tissue and cell integrity with concomitant

losses of immunostaining (e.g., due to proteolysis of the antigen). If acquiring animal tissues, consider

performing perfusion fixation before organ/tissue removal if it is an option

Steps

Tissue is harvested using our protocol (Tissue Harvesting Protocol)

We submerge the biopsy in the histology container prefilled with 10% NBF for fixation

Minimizing the warm ischemic time is critical. The time delay between tissue excision and fixation is detrimental to further analysis.

Note on the effect of delay to formalin fixation - Delay to formalin fixation effect on breast biomarkers - PubMed (nih.gov))

The rate of penetration of formaldehyde depends on the size of the biopsy. Trimming is often required to facilitate fixation. Tissues placed in the tissue cassettes should be no thicker than 3-4mm because the interior of a specimen may not become fully fixed, or significant autolysis can occur.

Reference - tmalab.jhmi.edu/histology.html#proc

Tissue Trimming

Trimming also helps remove unwanted parts of the biopsy such as excessive fat and traumatized

edges.

Label tissue cassette with sample ID with pencil

Place the trimmed tissue in cassette

Place cassettes back into the histology container

Tissue fixation (continue)

Refill 10% NBF into the container and leave for 12~48 hours at room temperature.

Use plenty of fixative. The general rule is to use at least 15 volume equivalents of formalin per volume

of tissue. A higher formalin-to-tissue ratio certainly won't hurt, and just requires a larger container.

Formalin is relatively cheap, so don’t skimp on this step.

10.

Gentle agitation of the tissue in the formalin during fixation maximize diffusion and reduce the poor local fixation.

11.

Pour the formalin into an “excess formalin” waste container in the hood.

12.

Rince the cassettes in the histology container with 1x PBS

13.

Drain and refill the PBS. Leave the cassette submerged in PBS and store at 4 degree C

until processing.

14.

Properly fixed tissue can be stored in PBS up to a week.

Tissue Fixation | HubMAP | JHU-TMC