I am working with CD68 immunohistochemistry on adipose tissue to detect macrophage in it. I fixed adipose tissue frozen sections in ice cold isopropanol for ten minutes, then after 2 hrs drying I used hot PBS with TWEEN to remove the fat from the vacuole of fat cell, then I followed the actual protocol for CD68 immunohistochemistry, would you please suggest me whether this hot PBS destroy the antogenicity of adipose tissue or not. Because I did not find any colour reaction in positive control. And I would like to get the standard protocol for CD68 IHC on adipose tisse macrophages. Any suggestion will be highly appreciated. Thanks in advance.
Sorry for any inconvenience.
My Email Address: firstname.lastname@example.org
I don't have specific information on whether your protocol could damage the CD68 epitope but I have found several protocols that don't use a heated buffer treatment. Here's one:
Immunohistochemical detection of macrophages was performed on frozen 8-eeCthick human adipose tissue cryostat sections and fixed in 4% paraformaldehyde for 5 min, followed by methanol for 5 min. The sections were blocked for endogenous peroxidase activity by incubation in 0.3% H2O2 in PBS overnight at 4°C. For CD68 immunostaining, mouse anti-human CD68 monoclonal antibody (clone EBM-11; Dako, Milan, Italy) was used at a dilution of 1:150 in blocking solution (ImmPRESS Reagent Anti mouse Ig; Vector Laboratories, Burlingame, CA) overnight at 4°C, followed by peroxidase-coupled anti-mouse IgG (ImmPRESS Reagent; Vector labs, Burlingame, CA) for 1 h at room temperature. (Diabetes August 2005 vol. 54 no. 8 2305-2313).
Adipose tissue, muscle, and liver samples were fixed for 12–16 hours at room temperature in zinc-formalin fixative (Anatech Ltd., Battle Creek, Michigan, USA) and embedded in paraffin. Five-micron sections cut at 50-µm intervals were mounted on charged glass slides, deparaffinized in xylene, and stained for expression of F4/80 as described by Cecchini et al. (43) with an anti-F4/80 monoclonal antibody provided by E. Richard Stanley (Albert Einstein College of Medicine), or for expression of CD68 with the commercially available monoclonal antibody PG-M1 according to the manufacturer’s instructions (Dako CytoMation, Carpinteria, California, USA). For each individual mouse adipose depot, four different high-power fields from each of four different sections were analyzed. The total number of nuclei and the number of nuclei of F4/80-expressing cells were counted for each field. The fraction of F4/80-expressing cells for each sample was calculated as the sum of the number of nuclei of F4/80-expressing cells divided by the total number of nuclei in sections of a sample. The same procedure was used to measure the fraction of CD68-expressing cells in human tissues. Adipocyte cross-sectional area was determined for each adipocyte in each field analyzed using image analysis software (SPOT version 3.3; Diagnostic Instruments Inc., Sterling Heights, Michigan, USA). Average adipocyte cross-sectional area was calculated for each animal using Microsoft Excel (Microsoft Corp., Redmond, Washington, USA). (J. Clin. Invest. 112(12): 1796-1808 (2003))
It could be that the solvent treatments (methanol or xylene) after fixation help to get rid of the fat.
Might be worth a try.