Frequently asked questions

• Got a good manual at hand ?
• Frozen or paraffin sections ?
• Immunohistochemistry (IHC) or immunofluorescence (IF) ?
• Which is the most sensitive method ? IF or IHC ?
• Alkaline Phospatase (AP) or Peroxidase (HRP) ?
• Which is the most sensitive, HRP or AP?
• Which is the most sensitive development system in HRP or AP?
• Do I need detergent to permeabilize the cells?
• For how long should I incubate a primary antibody?
• How stable are the diluted antibodies?
• Do I need to freeze my antibody aliquots?
• Why my slides do not stain today?
• Why only half of the section is stained?
• How can I reduce so much background?

FAQ: Frozen or paraffin sections ?
A:Some epitopes do not survive fixation and embedding.
From the same frozen block you can get DNA, RNA, free nuclei for FISH or cell cycle analysis, protein and a stained section on serial sectioning
From a paraffin block you can get DNA and RNA for PCR amplification (extensive crosslinking prevents extraction of long nucleotide stretches), free nuclei for ploidy and cell cycle analysis, cells for flow cytometry ^Ref and several section on serial sectioning.
Fixation and embedding prevents degradation and physical relocation of antigen (protein, DNA, RNA) by crosslinking.
Morphology on frozen is poor. Paraffin sections allow unlimited almost identical serial sections.
Cell and tissue shrinkage, associated with fixation and embedding, results in higher antigen density on the section.
Nasty bugs are destroyed by fixation
Most of the specimens stained in a routine immunohistochemistry lab in Surgical Pathology are formalin-fixed, paraffin embedded tissues. Fixation and embedding cause antigen masking, but also better retention of labile proteins, nucleic acids and small peptides. In order to overcome the drawback of antigen loss, enzymatic- or heat- mediated antigen retrieval is used (see a comprehensive ref. list here).

The methods detailed here, mostly tailored for paraffin sections, can be applied to frozen sections, cytospins, smears.
On such material, you may have to deal with significant presence of

endogenous enzymes (AP, AcPh, peroxidases)

endogenous biotin

Fc receptors

loss of cellular antigens and morphologic detail

diffusion artifact

You may not use antigen retrieval on frozen sections: there is no fixation and embedding-associated antigen masking. You may try heath-mediated quenching of endogenous phosphatases, but on crosslinking-fixed specimens only.

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FAQ: IHC or IF?
A:IHC allows you to see cytologic details and tissue architecture.
Preparations are permanent and relatively light-insensitive.
If you have very rare events, IHC allow you to screen large fields at low power.
Autofluorescence on some tissue makes IF very difficult.
IF gives quantitative signals (if you have a way to acquire signals quantitatively).
Co-staining the same subcellular structure with different fluorochrome is possible ONLY with IF.
Triple staining is possible only with IF.

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FAQ: Which is the most sensitive, IHC or IF?
A:IHC allows the greatest sensitivity because of a combination of reasons:

Excellent signal to background ratio, for high density antigens (>10K molecules / cell), with a simple indirect IHC.
An equivalent IF immunostaining is less sensitive because none of the fluorochrome for which filters are commonly available (except possibly Cy-3) matches the sensitivity and the signal-to-background ratio. Fluorochromes such as Pe or Cy-5 / APC, which are used in FCM and have very high quantum yield, either fade very rapidly (Pe) or have special filter requirements, not ususally found on epifluorescence microscopes..

For low density antigens (>2K,<10K molecules / cell), the only reliable and diffusely available signal amplification methods are in IHC.

NOTE: routine IHC or IF may  not detect antigen below 2K molecules / cell ^Ref. Flow cytometry sensitivity is approximately 1K molecules / cell and up.

FAQ: Alkaline Phospatase (AP) or Peroxidase (HRP) ?
A:Routine IHC is done with peroxidase (HRP) and DAB development, an insoluble, light insensitive, brown pigment which well contrasts with the blue nuclear counterstain of Hematoxylin. Can be mounted permanently with xylene-based mounting media, with excellent optical charateristics.
Red development of AP has even greater color contrast with Hematoxylin counterstain, however with most development, you should use an acqueous mounting.

Consider your tissue first:
hematopathology specimen are traditionally done with AP ^Ref, because of high endogenous peroxidase in BM and blood. Beware of cells with high endogenous Acid Phosphatase (AcPh; e.g. macrophages): if the pH of your development is off, you may stain endogenous AcPh along your exogenous AP.

If your tissue has levamisole-resistant endogenous AP (e.g. gut, bone), use HRP, unless you boil it (see below).

If your tissue has been exposed to high heath (MWO), your endogenous enzymes except HRP are gone ^Ref.

If your tissue has high endogenous peroxidase (e.g. BM), use AP.

If your tissue has endogenous biotin, barely quenched by blocking systems (e.g. kidney, liver), use a non-biotin-based system with AP.

 
You can use sodium azide (NaN₃)-containing solutions with AP-conjugates. You should not, whenever you have HRP.

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FAQ: Which is the most sensitive, HRP or AP? And which type of enzymatic development?
A:NBT/BCIP development of AP is by far the most sensitive type of development ^Ref, because both donor and acceptor substrate reagents precipitate in an insoluble very dark color. Furthermore, the reaction is not self-extinguishing, as in the case of DAB, by progressive burial of the reaction site by the reaction products.
NBT/BCIP is not widely used because is slow, does not allow good nuclear counterstain and cannot be mounted reliably in permanent mounting media. It also tends to diffuse if the section is thick.
Development of HRP with DAB and FastRed development of AP are the most preferred developments, because of speed, fine precise deposition, and great color contrast with nuclear hematoxilyn.
Sensitivity is also determined, in minor part, by the amount and degree of color contrast between the specific stain and the counterstain.

HRP-mediated deposition of Tyramide-conjugated molecules (biotin, fluorochromes) ^Ref is a powerful method to amplify the signal, followed by the development of choice (HRP, AP, fluorochrome).
Although very powerful, the methods is lenghty, and the final results can be appreciated only at the very end of the process, when no corrections can be done.

There are few studies comparing these methods and comparing the efficiency of each enzymatic development.

We have compared AP and HRP-mediated development of high density antigens detected by monoclonal antibodies, by serial dilutions of the primary antibody (Mouse IgG1 anti CD3 _z) on serial sections of formalin fixed, paraffin embedded, dewaxed, antigen-retrieved human tonsils.
We purposedly used simple indirect immunohistochemistry for all developments.
Development time was set to the maximum useful for each substrate, after which no further increase in staining is noted. NBT/BCIP was also incubated overnight (O/N)(three bottom right sections).


click on the image to enlarge

DAB and NBT/BCIP are clearly the stronger; however NBT/BCIP is able to detect molecules one log and half less abundant than any other development system.
Location and physical association of the antigen with subcellular structures (e.g. nuclear matrix or DNA) seems to influence the sensitivity of the detection, depending on the development you use.
Antigens such as RAG-1 or -2 ^Ref, Blimp-1 ^Ref, or CyclinD1 ^Ref are best visualized after combined heat and enzymatic (DNAse) retrieval, followed by NBT/BCIP development of AP or AEC development of HRP. Diazonium salt or DAB development, in our experience, has proven deleterious.

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FAQ: Do I need detergent to permeabilize the cells?
A:Once cells are fixed (in suspension, smeared on a slide, cytospun, inside a piece of tissue) they no longer can select intracellular traffic of molecules . In addition, processes such as drying and lipid solvent treatment (acetone, ethanol etc.) creates huge holes in the subcellular structure.
Antibodies can easily cross cell membranes (cytoplasmic, nuclear) in fixed cells. Whether they can reach the antigen in case of fixation dependent antigen masking, is another problem. No detergent is able to do antigen unmasking.
The crucial role of low levels of detergents such as Tween 20 is to reduce surface tension in washing solutions, allowing the slides to remain wet.
There is no role for detergent in permeabilization of fixed cells.
Only exceptions are:

saponin treatment of living cells for flow cytometry staining

high concentration detergent mediated extraction of nuclear / cellular matrix-associated substances in lightly fixed cells

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FAQ: For how long should I incubate a primary antibody?
A: First a short introduction to the problem. Each antibody is generated through a T-cell dependent B-cell activation, affinity maturation in germinal centers, isotype switch and selection. Selection is by choosing animal, immunization schedule, affinity purification, performance in a particular assay (Western Blot, ELISA, IF, flow cytometry, paraffin section staining), choice of clone if monoclonal, lot if polyclonal. You may have to use an antibody which has not been selected for the use you want.

With a monoclonal antibody, affinity constant for the antigen and crossreactivity with unrelated antigens are fixed values, being the immunoglobulin clonal. If you have a monoclonal with high affinity, as little as 5 minutes may be enough. If you know you are dealing with high-affinity antibodies, stick to a practical half an hour (± time for coffee). However, we noticed negative staining with strong antibodies incubated for as much as one hour, compared with overnight incubation (e.g. p27, BCL6). This may be due to a combination of factors (Ag density, masking, affinity).
We use almost invariably high dilutions and overnight incubations. If the antibody is crossreactive with an unrelated clone at low affinity, long incubation time may allow unwanted binding, but at this point chose another clone or a polyclonal antibody. Concentration does not play a significant role.

Polyclonal antibodies represent a gaussian curve of various species (clones) with diverse affinity and crossreactivities. The relative proportion of the most antigen-avid and crossreactive-free clones makes a good or a bad antibody.
You can play with time and dilutions: very short incubations and relatively high concentrations will favor high affinity clones, even if not the most representative species. Long incubations and high dilutions will favor the most prevalent species, even if with low affinity.

Other considerations besides affinity may play a role in your choice of primary antibody staining duration:

antigen density. You may want to incubate overnight for <5K molecules/cell.

masking. When you do double staining, the first stain implies some degree of non-specific masking.

convenience. For research purposes, you can split over two days your tedious work.

Secondary antibodies are usually selected for high specificity and high affinity, so 45 minutes are usually OK.

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FAQ: How stable are the diluted antibodies?
A:Antibodies diluted as directed (see Reagents & Solutions) are very stable. Months and probably years. One study ^Ref has sown extended shelf life for commercial reagents, beyond the manufacturer's expiration date. For antibodies used very diluted against low level antigens (<2K molecules/cell) is probably safer make fresh solutions monthly.

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FAQ: Do I need to freeze my antibody aliquots?
A:Each freezing cycle causes aggregation of the immunoglobulin and loss of titer. Avoid freezing aliquots that you expect to use out in the next couple of years. You may freeze reference reagents in small aliquots for reference or reagents to be stored without preservative.

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It is your responsability to troubleshoot your procedure by doing a scientifically correct experimental plan, setting up the controls (+ and -) and browse the datasheets, the literature and some good manual with the theory of IHC.

Don't miss the excellent manual from Dako: Handbook of Immunochemical Staining Methods.
This is a MUST READ item. (See here the historical 1983 edition).