Immunocytochemistry on Cytospun Slides Protocol

Protocol Search

Please enter a search word

Protocol Information


Title	Immunocytochemistry on Cytospun Slides
Source	Alison

Overview

The use of an immunoperoxidase procedure to localize different histologically important antigens and other markers was first introduced by Su-Ming Hsu and his associates. The patented procedure uses biotinylated antibody and a Avidin:Biotinylated enzyme complex, hence, it has been dubbed the “ABC” technique. This ABC technique can be used to stain tissues such as formalin-fixed, paraffin-embedded histological sections, or for the detection of proteins, DNA and RNA that have been transferred from gels (Western, Southern or Northern blots). In addition, the technique can be used with in situ hybridization to detect nucleic acid targets in cells and tissue sections by microscopy.

Procedure

Fix and permeablized cells on slides for 10 minutes in pre-chilled acetone.
Draw circles around samples on slide with a SUPER PAP PEN.
Incubate sections for 30 minutes in 0.3 H2O2 in methanol or water.
Wash slides for 5 minutes in buffer on shaker.
Incubate sample for 20 minutes in diluted normal serum from VECTASTAIN kit.
Wash slides for 10 minutes in buffer on shaker.
Incubate samples in primary antibody overnight at room temperature in a hydration chamber.
Wash slides 10 minutes on shaker.
Incubate sample for one hour in diluted biotinylated secondary antibody solution.
Wash slides for 5 minutes in buffer on shaker.
Incubate samples for 30 minutes with VECTASTAIN ABC Reagent.
Wash slides for 5 minutes on shaker.
Incubate the samples in DAB Substrate solution at room temperature until suitable staining develops.
Wash slides with tap water for a few minutes.
Incubate slides in 0.05 M sodium bicarbonate for 10 minutes, followed by blot.
Cover sample with DAB Enhancing Solution for 20 seconds and rinse quickly with tap water.
Dehydrate slides in increasing ethanol concentrations and clear with xylene.
Mount slides.
Cells are visible with a microscope at 400X and 630X.

Reagents/Solutions

Diluted normal serum prepared by adding 3 drops of stock serum to 10 mL of buffer. The preferred serum for blocking is prepared from the same species in which the biotinylated secondary antibody is made.
DAB (3,3'-Diaminobenzidine tetrahydrochloride ) Substrate solution prepared by adding 2 drops of Buffer Stock Solution to 5 mL of distilled water and mixing well. 4 drops of DAB Stock Solution is then added and the solution is mixed again. Finally 2 drops of Hydrogen Peroxide Solution is added and the solution is mixed again.
PBS Buffer (10 mM sodium phosphate, 0.9% sodium chloride, 0.1% Triton X-100, pH 7.5)
Primary antibody typically prepared at a 50 X dilution in normal serum.
Diluted secondary antibody prepared by adding 50 uL of stock antibody to 10 mL of buffer.
VECTASTAIN ABC Reagent prepared by adding 100 uL of Reagent A to 10 mL of buffer, then adding 100 uL of Reagent B and mixing immediately. Solution should sit 30 minutes before use.

References

Am. J. Clin. Pathol., 75, 734 (1981); J. Histochem. Cytochem., 29, 577 (1981).
Kelleher AL (2003) Investigation of the mechanism for exclusion of p53 from the nucleus of leukemic cells of the soft-celled clam, Mya arenaria. MS thesis. University of New Hampshire
Vector Labs Troubleshooting for VECTASTAIN http://www.vectorlabs.com/Protocols/Supprotocols/tsg.pdf

Tips

Fixing of slides in acetone and all wash steps with buffer can be done in Coplin jars.
15-30 minutes is sometimes needed for suitable staining to be seen, but sometimes 2-10 minutes will suffice.
Nickel solution was not used in my procedure as it caused a clumping reaction upon application of the slide.
Be cautious when using the DAB solution, as it is a suspected carcinogen.
5-20 seconds may be sufficient to develop in DAB Enhancing Solution.
It is best to mount slides with an adhesive that will permanently attach the coverslip.