Immunohistochemical detection of cytokines in paraffin-embedded mouse tissues.
We have successfully developed a method for the immunohistochemical detection of interleukin 2 (IL-2), IL-4, IL-6, IL-10O, IFNgamma and TNFalpha using monoclonal antibodies (MAb), in sections of mouse tissue embedded in paraffin wax. The method involved fixation in periodate-lysine-paraformaldehyde (PLP), rapid dehydration and infiltration under vacuum with paraffin wax at 54 degrees C. Comparative observations demonstrated that the method gives equivalent or better results than formaldehyde fixed, frozen sections. Since reliable controls, both positive and negative, are paramount for interpretation of immunohistochemical staining, such controls were determined. The following tissues were shown to be suitable as positive controls when using paraffin-embedding: spleen for the detection of TNFalpha, small intestine for IL-2, IL-4 and IL-10, and HSV-1 infected eyes for IL-6 and IFNgamma. We conclude that PLP fixation and low temperature paraffin-embedding is a method which provides both preservation of excellent tissue morphology and reliable immunohistochemical identification of cytokines. These attributes will be invaluable in a wide variety of experimental situations.
Whiteland
J L
JL
Department of Ophthalmology, University of Bristol, UK.
Shimeld
C
C
Nicholls
S M
SM
Easty
D L
DL
Williams
N A
NA
Hill
T J
TJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015925:Cryopreservation; D016207:Cytokines / Q000032:analysis*; D005404:Fixatives; D005557:Formaldehyde; D007124:Immunoenzyme Techniques; D007132:Immunoglobulin Isotypes / Q000276:immunology; D008239:Lysine; D051379:Mice; D016612:Paraffin Embedding; D010504:Periodic Acid; D051381:Rats; D013194:Staining and Labeling
D000911:Antibodies, Monoclonal; D016207:Cytokines; D005404:Fixatives; D007132:Immunoglobulin Isotypes; C046311:periodate-lysine-paraformaldehyde; D010504:Periodic Acid; D005557:Formaldehyde; D008239:Lysine