Feb 14, 2025
Fixation & Tissue Processing Protocol
- Roy Ambli Dalmo1
- 1The Arctic University of Norway
Protocol Citation: Roy Ambli Dalmo 2025. Fixation & Tissue Processing Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6dzy1vqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 13, 2025
Last Modified: February 14, 2025
Protocol Integer ID: 120172
Abstract
Fixation & Tissue Processing Protocol
Fixative
Fixative
Note
* Freshly made RNase-free 4% paraformaldehyde (4% PF) used specifically for In Situ Hybridization
* 4% paraformaldehyde or 10% buffered formalin used for routine histology (i.e., H&E staining)
* 2% paraformaldehyde (bgal, GFP, RFP), 4% paraformaldehyde or 10% buffered formalin used for Immunocytochemistry
Fixation procedure
Fixation procedure
Note
The following protocols are for tissue sample processing for subsequent light microscopy with H&E staining, immunocytochemistry or in situ hybridization. RNase-free PBS and fixative should be used for in situ hybridization with RNA probes.
* Use the 20 ml scintillation vials with 15-20 ml solution volumes or
60 ml yellow-capped containers with 30-40 ml solution volumes to fix, wash and dehydrate the samples.
Well-fixed samples can be stored in the same fixative at 4˚C for several days or even longer for H&E staining. In situ hybridization samples must be dehydrated and stored at -20 oC with 100% ethanol after 24-48 hours fixation. Immunohistochemistry samples must be dehydrated and stored at -20 oC with 100% ethanol after overnight fixation.
Tissue fixation
Tissue fixation
Isolate the tissue into cold PBS as soon as possible (can be omitted).
Wash tissue with PBS to remove all blood (can be omitted).
Place tissue in fixative for 10-15 minutes to one hour (can be omitted)
Cut tissue to proper size. The size can be 2X2 mm to 1X2 cm but thickness should be 3 mm for better fixation. The cutting surface of the tissue should be flat and smooth.
Transfer tissue to fixative and swirl the container to ensure all tissues are completely immersed in fixative. The volume of fixative must be 20-30 times the tissue volume.
Fix tissue at 4˚C for overnight.
Check sample the next day to ensure proper fixation.
For best morphology and staining results take care to:
For best morphology and staining results take care to:
Note
1. Use high-quality fixative and sufficient amounts of fixative.
2. Use sharp razor blade and fine instruments such as scissors and forceps to handle all specimens. A piece of dental wax can be used as a cutting board. Treat tissues as gently as possible to minimize morphology artifacts.
3. Don’t let samples dry during preparation. Samples should always be covered by solutions.