You just can’t put raw tissue or cell samples on your slides and expect good histology results! Instead you must preserve or ‘fix’ your samples. Fixing ensures that your cell structures stay intact and that your antigens are immobilized. Ideally, fixation would also still permit unfettered access of your antibodies to your antigens. However, as you will learn in this primer, this is often a game of give and take. The better your tissue is preserved the more inaccessible your antigens become.
To optimize your fixation methods for both preservation and antigen availability, you will need to experiment. But hey, that is what you are (poorly!) paid for- experimenting! So roll up your sleeves, read on to learn more about fixation, and perfect your fixation protocol.
There are two types of fixation
There are two common classes of fixation: (1) Organic solvent methods and (2) The cross-linking method. The goal of both methods is to denature your proteins and thus stop your samples from decaying. To do this, the organic solvent methods use…wait for it… organic solvents (bet you didn’t see that coming!) such as various alcohols or acetone. Alcohol or acetone works to preserve your samples by removing lipids, dehydrating your tissue, and denaturing and precipitating the proteins in your samples.
In contrast, the cross-linking method uses paraformaldehyde to form covalent chemical bonds (or cross-links) between the proteins in your tissue and their surroundings. This method anchors proteins within the cytoskeleton of the cell and in the process imparts rigidity to your samples. The rigidity provided by the cross-linking method is a bonus when sectioning fragile or porous tissues.
Each method has its good and bad points
In general, the cross-linking method better preserves tissue integrity and subcellular structure than the organic solvent methods. Therefore, if you can, you should to use the cross-linking method. However, sometimes you may not be able to use the cross-linking method.
Cross-linking is usually best…but not always
Immunohistochemistry uses antibodies which recognize specific antigens in tissues. Cross-linking can ‘mask’ these antigens. If your antigens are ‘masked’ it means that the cross-linking (or other fixation method) is physically blocking or otherwise preventing your antibody from recognizing your antigens. Not good. Luckily this can often be reversed. There are numerous methods to ‘unmask your antigens’. These methods usually use some combination of proteinases, heat and chelators to make your tissues more easily penetrated by your antibodies. For a full article on unmasking your antigens see Catriona’s article on antigen retrieval.
There is no way to anticipate the best fixation/unmasking methods for your immunohistochemistry. Instead, you will need to test a variety of conditions.
But before you start optimizing your fixation method here are a few tips:
Tips before you start your protocol optimization;
Tip #1: Select an appropriate troubleshooting tissue sample. Use cells or tissue samples that are as similar as possible to your final samples. However, save your best and most valuable samples until you have completed your histology troubleshooting and have established a good, consistent protocol. For example, if you are interested in the different staining patterns between wildtype and knockout mouse livers, heterozygous mouse livers might be a great tissue to troubleshoot with.
Tip#2: Fix at the right time and place. Tissue samples should be fixed immediately following harvesting. Fix tissue samples by fully submerging your tissue samples in labelled eppendorf tube or conical tube containing fixation solution (See Table 2 for fixation recipes). Larger tissue samples should be cut into 2 mm blocks before fixing. Conversely, single cells, such as from cell culture, should be attached to your microscope slides before you fix. How to attach and fix cells to your microscope slide will be addressed later this month.
Tip #3: Know (do not assume) that your tissue expresses your protein of interest. Please, please, please, before you do any immunohistochemistry troubleshooting, run a Western blot with your tissue and antibody in question to ensure that your tissue does express your protein-of-interest. This double check can save you a lot of time trying to perfect an impossible immunohistochemistry protocol!
How to Optimize Your Fixation Method
If you are establishing a de novo immunohistochemistry protocol with an untested antibody, I suggest that you start with at least eight fixation/unmasking experiments- see Tables 1 & 2. These eight experiments will allow you to compare an organic solvent fixing method (you can use either recipe #1 or #2 from Table 2, or another favorite) against a basic cross-linking fixing method with and without various unmasking techniques.
Table 1: Fixation/Unmasking Optimization Scheme.
|# 1||An Organic Solvent Method||skip||Stain normally||+ control for organic solvent method|
|# 2||An Organic Solvent Method||skip||no secondary antibody||– control for organic solvent|
|# 3||Cross-Link||skip||Stain normally||+ control for cross-linking|
|# 4||Cross-Link||skip||no secondary antibody||– control for cross-linking|
|# 5||Cross-Link||Unmask with Tris-EDTA and heat||Stain normally||+ control for cross-linking and EDTA unmasking|
|# 6||Cross-Link||Unmask with Tris-EDTA and heat||no secondary antibody||– control for cross-linking and EDTA unmasking|
|# 7||Cross-Link||Unmask with Digestion||Stain normally||+ control for cross-linking and enzyme unmasking|
|# 8||Cross-Link||Unmask with Digestion||no secondary antibody||– control for cross-linking and enzyme unmasking|
Note: Do not attempt to cut corners and leave out your negative staining controls in these experiments! Various fixation and unmasking conditions can affect your level of non-specific background staining, therefore it is important to have negative controls.
Table 2: Detailed Fixation and Unmasking Methods
|Fixation Methods||Organic Solvent Method #1||Use -20°C Acetone, or Methanol, or a 1:1 Acetone/Methanol mix to fix isolated cells for 5-10 minutes. For larger tissue samples fix for an hour or more. Rinse a few times with PBS.|
|Organic Solvent Method #2||Use -20°C 95% ethanol and 5% glacial acetic acid solution to fix isolated cells for 5-10 minutes. For larger tissue samples fix for an hour or more. Rinse a few times with PBS.|
|Cross-Linking Method||Use 3-4% paraformaldehyde to fix isolated cells for 5-10 minutes. For larger tissue samples fix for up to 30 minutes. Rinse a few times with PBS.|
|Unmasking Methods||Tris-EDTA & Heat Unmasking Method||After your cells or tissue is fixed and attached to your slides, fully submerge your slides in a 10 mM Tris Base, 1 mM EDTA, 0.05% Tween 20, pH 9 solution for 10-40 minutes at 95-100?C. When done allow your slides to fully cool (20 minutes). Rinse a few times with PBS before proceeding to staining steps.|
|Digestion Unmasking Method||After your cells or tissue is fixed and attached to your slides, fully submerge your slides in 20 g/ml solution of Proteinase K in TE buffer, pH 8 for a 10-20 minute incubation at 37?C. Rinse a few times with PBS before proceeding to staining steps.|
When analyzing the results from your fixation optimization experiment, you are looking for the fixation method that gives you;
(1) Good tissue preservation and,
(2) A strong specific stain.
When I say a “good specific stain”, I mean that you should get strong staining results under normal staining conditions (+ control) and weak (or no) staining results when you leave out the secondary antibody (- control).
There is a good chance that in the four conditions tested in Table 1 you will find a workable fixation method. However, as you will learn (or have already painfully learned) histology often takes a lot of trial and error to find what works best. If none of these initial four permutations are showing good specific staining patterns and tissue preservation, you may need to try other fixation or unmasking combinations, or troubleshoot your antibody incubation concentrations and times.
Good luck and happy fixing!