Standard Chemical Tissue Fixation
Aldehyde fixatives (formalin, 10% Neutral Buffered Formalin (NBF), 4% Paraformaldehyde (PFA), gluteraldehyde) “fix” tissue (i.e., arrest biological degradation) by forming chemical “cross-links” between and within proteins. The most commonly used is NBF, which is purchased in a ready-to-use form and is thus very convenient. Arguably, 4% paraformaldehyde is, in many cases, a superior fix for immunohistochemistry as it tends to yield reduced background staining but must be freshly made just before use.
Many researchers believe that tissue may be stored indefinitely in 10% NBF without harm. This is partly true; sections cut from such material and stained with a routine histological stain (e.g., Hematoxylin and Eosin-‘H&E’) will look fine. Problems, however, will likely arise when such material is used for immunostaining since the cross-linking action continues and may irreversibly change the conformation (i.e., reduce the antigenicity) of antibody targets. If you intend to use your tissue for immuno-staining be careful not to ‘over-fix’ the tissue. Fixation method / time / temperature is a variable that needs to be considered when developing immunohistochemistry protocols. Significantly altering fixation times between batches of specimens can have major consequences on the quality of immuno stains.
Some general suggested fixation guidelines:
For routine fixation in 10% NBF, dissect the tissue as rapidly as possible and immerse in a large volume of fix (i.e., at least 20X volume of fix to tissue). Place at 4 degrees and fix ‘overnight’. After fixation, wash the tissue well in several changes of Phosphate Buffered Saline (PBS). Tissue may be stored in cold PBS for short periods of time (2 or 3 days) but should be placed in 70% ethanol for extended storage. The tissue will be safe in the EtOH since there is no danger of bacterial degradation. However note that it has been reported that proteins can be extracted by extended storage in ethanol and that storage in PBS with sodium azide added at 4 degrees is a better choice. Safest of all is to promptly fix, wash and deliver your samples for processing and embedding. Tissue may be brought to the Morphology Core for processing in either PBS or 70% EtOH.
The size of the specimen being fixed should be considered. A commonly quoted rate of penetration for aldehyde fixes is two-to-three millimeter per hour. This rate is, of course, variable depending on the density and other characteristics of different tissues and is simply a rule of thumb, but it follows that trying to fix very large pieces of tissue runs the risk of under-fixing the interior of the specimen resulting in biologic degradation of the constituent molecules. For solid material (e.g., liver) the longest dimension should not exceed 10-15mm.
For very small pieces of tissue (~1-2mm range dimensions) an overnight fix may in fact be too long. One to several hours may be a better choice for this material but be aware that changing times of fixation between experiments may have serious effects on subsequent immunostains. Please try to be consistent to reduce variable staining results.
Some specific considerations:
1) Different tissues degrade at different rates post mortem. When collecting multiple organs it is wise to fix the enzyme-rich ones first. For example, pancreas degrades extremely rapidly and should always be taken first followed by liver and so forth.
2) Tissue hardens upon fixation and remains in whatever shape it was fixed in. This should be considered in those cases where the final orientation of the sections is important. Pancreas, for example, is very pliable when dissected and if simply dropped into fix will likely harden into some rough spheroid shape. If your intention is to look at sections with a “maximum footprint” (a term of art meaning maximum viewable area) the pancreas has to be spread out flat in a cassette lined with a biopsy sponge before fixation occurs.
3) The quality of sections cut on a microtome suffer badly from several (avoidable) causes. During the post fixation processing step all water is extracted from the cellular spaces of the tissue and is replaced with paraffin. This is carried out using a vacuum-based automatic machine in which the tissue is dehydrated via a series of graded ethanols followed by xylenes and finally liquid paraffin. The processed tissue is then oriented in a mold, embedded in a paraffin block and is ready to section. The density and general consistency of the tissue is more or less that of the paraffin and cutting good sections is fairly straightforward. The inclusion of materials with radically different hardness in the block, however, wrecks havoc! When such material comes in contact with the microtome blade it causes severe ripping and tearing of the entire section.
Things to avoid include: fecal material in intestine, especially in the colon where this material is very hard; hair is particularly bad—if you are bringing us mouse skin to be sectioned be sure to remove the hair before fixation. This can be done using a razor blade or clippers. The Morphology Core will loan you our clippers if you need them. Hair can also sometimes be inadvertently included with organs. Please be careful during dissections; sutures-if you have done a ligation prior to dissection please be sure to not include the thread or staples with the specimen.
4) Labeling tissue cassettes. The cassettes into which the tissue is placed are subsequently exposed to ethanols and xylenes in the processor. If you label your cassettes with a regular magic marker the writing will come off in the xylene. It is devastating to lose the identity of your specimens! The Morph Core is aware of this and we are generally suspicious of pre-labeled cassettes. We try to test the labeling for xylene insolubility before processing but why take the chance that we may forget to do this? Unless you have the special pens we use, label your cassettes with pencil!
Cryopreservation
There are several good reasons to use cryopreservation rather than chemical fixation that include: minimal alteration of the structure of proteins to be identified by immunostaining; to avoid / reduce the ‘auto fluorescence’ (i.e., high background) that is inherent to formalin fixed paraffin embedded (FFPE) tissue when performing immunofluorescent microscopy; the preservation of delicate ‘reporter’ enzymes (e.g., beta gal, GFP) that are easily destroyed by fixation; the need to do a lipid stain (e.g., Oil Red O) requires frozen sections since the ethanols used in tissue processing will extract the lipids. The morphology of cryo sections, however, is often inferior to paraffin sections.
General suggested cryopreservation guidelines:
Essentially there are two ways to freeze your tissue—Fresh-frozen and Fixed-frozen.
Fresh Frozen: Tissue is dissected and frozen as rapidly as possible. The Morphology Core has a “snap-freezer” (marketed as a ‘Gentle Jane’) that is simple to use and produces excellent results. This device may be borrowed from the Core. The tissue is stored at -80 until ready for sectioning. Frozen slides are warmed to room temperature for approximately 10 minutes and then briefly ‘post-fixed’ prior to staining.
Fixed-Frozen: Tissue is formalin-fixed in the usual manner followed by washes in PBS. The tissue is then ‘cryo-protected’ (water displaced from the cellular spaces) with sucrose. There are many variations on the sucrose infiltration step. We suggest that you simply place the fixed tissue in a 30% sucrose solution (in PBS) at 4 degrees and wait for it to sink (the water has been displaced when the tissue reaches negative buoyancy). All excess sucrose solution must be then blotted from the exterior of the tissue prior to embedding it in OCT and freezing it with the snap freezer. This method is useful when exposure to the ethanols and xylenes used in FFPE processing needs to be avoided.
Other Fixes
Carnoy's (coagulative / precipitating fix-may be useful for collagens, actin, keratins, gfap, etc.)
Ethanol: chloroform: acetic acid (60:30:10 v/v)
Methacarn (coagulative / precipitating fix-may be useful for collagens, actin, keratins, gfap, etc.)
Methanol : chloroform : acetic acid (60:30:10 v/v)
Bouins
Saturated aqueous picric acid : formalin : acetic acid (15:5:1 v/v)
Antigen Retrieval for Immuno-staining
The protein cross-linking of formalin fixation described above often inhibits antibody binding due to conformational changes. There are a number of methods commonly used to restore antigenicity. These methods are not generally used when working with cryo-preserved sections.
Microwaving. Microwave antigen retrieval is one of several so-called ‘HIER’ (heat induced epitope retrieval) methods. Others include steaming, pressure cookers and autoclaves. Typically this type of retrieval involves boiling the slides in a pH 6 citric acid buffer for ten minutes, followed by an additional fifteen minutes in the hot buffer (this is the Morphology Core ‘default’ method). There are instances where changing the pH to 3 (in citric acid buffer) or to 8-9 (in a Tris EDTA buffer) are successful. Please see Protocols.
Enzymatic: Partial digestion of tissue sections with one (or several) enzymes can sometimes be effective. Protocols are provided for pepsin, protease and trypsin. The enzyme concentrations and incubation times given may have to be adjusted.
Immuno -histochemistry & -fluorescence
Please consider the following:
The antigenicity of tissue sections on slides can be reduced or eliminated by long term storage (Blind, C, et al J Clin Pathol 2008;61:79–83; Fergenbaum, JH, et al Cancer Epidemiol Biomarkers Prev 2004;13(4). April 2004). It is generally held that target proteins can be oxidized at ambient conditions. There is heterogeneity of this phenomenon: some targets are very susceptible and others are largely unaffected. A variety of storage methods have been suggested to counter this including keeping the slides at reduced temperatures (4°, -20°, -80°), desiccation, vacuum sealing, storage under nitrogen, protection from light and 'sealing' the sections with paraffin. To reduce the chance of false-negatives, we suggest that you avoid having large numbers of slides cut weeks or months ahead of doing your immuno-staining experiments.
Formalin-fixed-paraffin-embedded (FFPE) tissue sections typically 'autofluoresce' when viewed under a fluorescent microscope (some tissues are worse than others); for this reason, immunofluorescensent (IF) staining is best done on frozen sections. However, we routinely perform successful IF on FFPE sections (as long as a good signal-to-noise ratio can be generated). Background autofluorescence, to a greater or lesser degree, can be suppressed by a post-staining Sudan Black treatment. Using a far-red fluorophore (e.g., Cy 5) can also be helpful since there is little autofluorescence at these wavelengths. Moderate background can be removed from digital images.
Do not reuse / save diluted antibodies. The antibodies adhere to the walls of the container and this effectively reduces the titer to zero.
Do not subject antibodies to freeze / thaw cycles, it destroys them.
'Dirty' (high background) polyclonal antibodies can sometimes be 'cleaned up' by storing the antibody over pieces of fixed tissue. Irrelevant antibodies in the serum may be adsorbed onto the tissue, effectively raising the titer of the specific antibodies. Another way to approach this is to incubate the antibody on a tissue section and then reuse the antibody on a second section.
Suggested work-up of antibodies.
For new antibodies we will typically run three dilutions (e.g., 1:100, 1:500, 1:1000) and a no-primary, secondary only negative control on two sets of slides (one with antigen retrieval, the other without) using our default staining protocol. Results are compared with expected / published staining patterns.
If the stain looks good, additional controls are done in subsequent experiments (e.g., inclusion of biologically negative tissues, antigen (peptide) blocking, non- or pre-immune serum, isotype controls). Additional titration of the antibody is also done.
If the staining is poor, our next step, generally, is to adjust the pH of the antigen retrieval buffer and repeat the stain on two more sets of slides, one at pH3 and he other at pH8.
Still poor? Try enzyme retrieval
Still poor? Try alternate fixes and or frozen sections
Still poor? Buy a different antibody.
Standard (pH6) Immunohistochemistry Protocol for
Formalin-fixed-paraffin-embedded tissue
University of Pennsylvania
Department of Medicine / Gastroenterology Division
Center for Molecular Studies of Digestive and Liver Diseases
Morphology Core
I) Tissue Fixation
1) Put tissue into a good volume of (either)
4% paraformaldehyde (in 1X PBS) or 10% Neutral Buffered Formalin O/N at 4°
2) Wash tissue in 1X PBS 30’ to several days at 4°
3) Process and block in paraffin
II) Pretreatment of sections:
1) Dewax tissue sections:
Warm slides at 6015’
Xylene 2 X 5’
100% EtOH 2 X 3’
95%, 90%, 80%, 70%; 1’ each
Water
2) Immerse slides (in plastic slide rack) in 10mM citric acid buffer (Fisher cat.# A104-500; Citric Acid Monohydrate) (pH 6.0) and incubate in microwave oven. Microwave on high. 15'
3) Remove from microwave and let stand in the hot buffer15’
4) Rinse in gently-running tap water5’
5) Quench endogenous peroxidases: immerse slides in
ddHOH (185ml) / 30% hydrogen peroxide (15ml) 15’
6) Rinse in gently-running tap water 5’
7) Lay slides out in hybridization box. Wash 1X PBS 5'
(Use hydrophobic pen to restrict subsequent volumes of
reagents applied to the sections).
8) Block with Avidin D blocking reagent (Vecta) at RT15’
Note: Avidin reagent must precede biotin reagent.
9) Quick rinse with PBS
10) Block with Biotin blocking reagent (Vecta) at RT15’
11) Quick rinse with PBS
12) Block with Protein Blocking Agent 10-15’
(StartingBlock™T20 (PBS) Blocking Buffer Thermo Scientific #37539).
(alternate block: 5-10% normal serum from host species of the secondary antibody 30' at room temperature)
13) DO NOT rinse, tip excess blocker off slide and proceed
with Ab immunohistochemistry
III) Immunohistochemistry (using HRP as reporter molecule)
1) Primary antibody
dilute 1:XX ('optimal' dilution) in PBT1. (use water-soaked paper towels
to create a moist atmosphere in the hybridization box). Overnight at 4°
2) Rinse and then wash in PBS 2 X 5'.
IV) Apply Secondary antibody. (Vector Laboratories; anti (species of primary host),
dilute 1:200 in PBT
1) Incubate at 37° 30 minutes
(Be sure to make the HRP-ABC solution from the kit at this time since it needs to be combined for at least 1/2 hour prior to use; longer times are also OK).
2) Rinse and then wash in PBS 2 X 5'.
V) Apply HRP-conjugated ABC reagent from Vecta ‘Elite’kit (10µl reagent A and 10µl reagent B per ml PBT).
1) Incubate at 37° 30 minutes
2) Rinse and then wash in PBS 2 X 5'.
VI) Develop signal (use Vector Laboratories ‘DAB Substrate Kit for Peroxidase’)
(Note-make immediately before use)
1) Per 2.5ml of distilled water: add one drop of Buffer Stock Solution. Mix well. Add two drops DAB Stock Solution. Mix well. Add one drop Hydrogen Peroxide Solution. Mix well. Wear gloves. Collect waste for chemical disposal.
2) Apply this solution at room temperature and monitor signal
development in dissecting scope (stop reaction when a good signal-to-noise ratio is achieved. Excessive development only leads to background) 30”-10’
3) Wash sections for 5+’ in distilled water
VII) Counter stain with Gills #2 Haematoxylin (Fisher) / dehydrate and mount.
1) Immerse very quickly in Haematoxylin and rinse quickly with water. Inspect results in microscope. Try to achieve a good balance between DAB reaction product and hematoxylin i.e., light brown / light blue, dark brown / dark blue. It is easy to add more hematoxylin by re-dipping. If you overstain, remove (some) hematoxylin by dipping slides in 0.5% acid alcohol (0.5ml HCL / 99.5ml 70% ethanol).
2) Rinse with water
3) 70% EtOH 2’
4) 95% EtOH 2’
5) 100% EtOH 2 x 2’
6) Clear in Xylene2 x 1’
7) Mount
1PBT per 50ml
H2O43.5ml
10X PBS 5.0ml[1X]
10% BSA 0.5ml[0.1%]
10% Triton X-100 1.0ml[0.2%]
Links of Interest:
(It is interesting to consider the many opinions expressed regarding the must "does and don'ts" of antibody staining. Essentially, all IHC / IF protocols are variations on a theme. The only universal 'truths' in this business are that a) no single optimal fixative, antigen retrieval method, or staining condition exists and b) immunostaining is the essence of empiricism.)
Histology / Immunohistochemistry
Microscopy