A user note for C023, C024, C025, C026, C027.
Sections from L.R. White embedded tissue have been used successfully for immunocytochemistry at both the light microscope and electron microscope levels. This demonstrates quite clearly that the visualisation steps of the immunocytochemical procedure will penetrate the resin and react with the tissue antigens if they have been preserved in the tissue.
As with all immuno‐localisations, the key factor is whether or not the tissue antigen has survived fixation, processing and embedding in such a form as to be recognisable to the specific antibody. This is difficult to predict with certainly, but some antigens have been shown to be highly resistant whilst others are fickle even in unfixed frozen sections of antisera Much interest has centered on using immunocytochemistry to detect protein hormones and the various classes of immunoglobulin and generally these classes of antigen have proved resistant to alteration both in processing to paraffin‐wax and to L.R. White.
It is the special hydrophilic nature of L.R. White which allows immunochemicals to permeate the supporting resin and reach its sites of binding and no resin pretreatment is necessary, or indeed possible, to facilitate this penetration.
We have been successful with L.R. White blocks only when they have been thermally cured, probably because when accelerator‐curing the resin the exotherm produced is sufficient to damage the integrity of the tissue antigen. Some workers have also reported that a slight under‐cure of L.R. White, say at 55 degrees C for 20‐24 hours, aids subsequent penetration, but we have obtained good results without deviating from the standard polymerisation schedule.
If the particular antigen under consideration has already been localised in paraffin‐wax sections then a trusted fixation regime will be established and should be adhered to. For those approaching the problem for the first time there is an extensive bibliography available regarding fixation for immunocytochemistry, much of it contradictory, and a reference list is provided as some guidance. Rules of thumb seem to be to avoid glutaraldehyde and perhaps use an acid rather than a neutral fixative, but there are many conflicting and strongly held views on the topic.
Similarly, the need to enzyme digest sections prior to reaction is fraught with controversy and may indeed be linked to the fixation regime chosen. We have used both protease type VII and trypsin type II to good effect on our neutral buffered formalin fixed material.
If frozen, dewaxed or etched epoxy resin sections are used for immuno‐staining, the tissue is not surrounded by a supporting matrix when they are being reacted. When using L.R. White sections the resin is still intact and therefore diffusion to the sites of reaction must occur prior to reaction of antisera with antigen. For this reason we have found it necessary to use antisera at approximately ten times the concentration that would work on de‐waxed sections. The exact titre of each antibody will depend upon its source and how well it has been stored, but we have used many commercial anti‐immunoglobulins at about a 1 in 10 dilution.
For the same reason the antibody stages of the reaction often benefit from a longer incubation time. Up to 2 hours at room temperature or overnight at 4 degrees C in a moist chamber may be used.
Various immunoperoxidase techniques have given results on L.R. White tissue sections including the peroxidase‐antiperoxidase complex method (PAP) (Sternberger, 1970), the hapten sandwich technique (Jasani et al, 1981), and the indirect peroxidase method. The Avidin‐biotin‐peroxidase complex method of Hsu has not been successful in our hands with L.R. White embedding material, probably due to the molecular size of Avidin. As fairly strong antibody concentrations are required, a highly sensitive method of detection is to be preferred and for this reason the PAP or hapten sandwich techniques are more suitable than a two layer indirect peroxidase reaction. Visualisation of the bound peroxidase is achieved with the diaminobenzidine‐peroxide reaction as described by Graham and Karnovsky (1966).
Any technique where the sections are subjected to hydrogen peroxide solutions twice during staining is likely to tend to lift sections from the slides. We have found that lysine (MW 350,000) is an excellent adhesive for immunocytochemical work, and also care should be taken to dry sections onto slides very thoroughly in an oven rather than a hot plate at 60 degrees C for two hours. This step should not have any effect on the antigenicity of the tissue as it will already have spent 20‐24 hours at 60 degrees C during polymerisation.
It is clear that no 'standard' immunohistochemical staining regime can be cited as there are so many variables, but a 'typical' regime is described below for general guidance.
Pap Procedure for L.R. White Sections (3 micron)
- Block Endogenous Peroxidase with 1% Phenylhydrazine Hydrochloride in P.B.S. (optional) 30 minutes
- Wash in P.B.S 1 x 5 min, and 2 x 5 mins at 37 degrees C
- 0.1% Trypson in 0.1% CaCl2 (aqueous) 20 minutes
- Wash in ice cold distilled water 10 minute
- 2% Goat serum in PBS 20 minutes
- 1° Antibody (approx 1:10 dilution) 2 hrs at 37 degrees C or overnight at 4 degrees C (usually Daco or Nordic)
- Wash in P.B.S. 10 minutes
- Goat anti rabbit antibody (approx 1:20 dilution) 2 hrs at 37 degrees C or overnight at 4 degrees C
- Wash in PBS 10 minutes
- PAP at 1:10 dilution (Dako) 2 hrs at 37 degrees C or overnight at 4 degrees C
- Wash in PBS 10 minutes
- Wash in Tris HCL pH 7.6 10 minutes
- DAB/H2O2 (Graham and Karnovsky) 15 minutes
- Wash in distilled water.
- Counterstain as required
NOTE: FOR IMMUNOCYTOCHEMISTRY L.R.WHITE MUST BE THERMALLY CURED AND NOT ACCELERATOR CURED
- CURRAN, R.C.and GREGORY, J., The unmasking of antigens in paraffin sections by Trypsin. Experientia, 33; 1400 (1977)
- ELIAS, J.M., Principles and Techniques in Diagnostic Histopathology. Published by Noyes Publications New Jersey, USA (1982) ISBN‐0‐8155‐0903‐0.
- GRAHAM, R.C.and KARNOVSKY, M.J., The early stages of absorption of injected horseradish peroxidase in the procimal tubules of mouse kidney: ultrastructural cytochemisty by a new technique. J. Histochem. Cytochem, 14, 291 (1966)
- JASANI, B., WYNFORD‐THOMAS, D.,and WILLIAMS, E.D., Use of monoclonal anti‐hapten antibodies for immunolocalization of tissue antigens. J. Clin. Path. 34, 1000 (1981).
- NEWMAN, G.R., JASANI, B., and WILLIAMS, E.D., The preservation of ultrastructure and antigenicity, J. Microscopy, 127, RP5‐RP6 (1982).
- STERNBERGER, L.A., HARDY, P.H. Jr., CUCULIS, J.J. and MEYER, H.G., The unlabelled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigen‐antibody complex (horseradish peroxidase‐antihorseradish peroxidase) and its use in identification of spirochetes. J. Histochem. Cytochem., 18, 315 (1970)