Pressure cooker

Antibodies were tested for reactivity on a variety of human reproductive tract tissues (cauda epididymis, vas deferens, vagina) and tonsil. Paraffin-embedded tissues were sectioned at 5 µm, de-waxed, hydrated, and subjected to antigen retrieval which was carried out by immersing the sections in a citrate buffer, pH 6, and then placing the tissues in a pressure cooker that was heated to 125°C for 30 seconds (Biocare Medical, Concord, CA). The sections were incubated with Cy3-labeled HCA or VRC01 (conjugation kit: Abcam, Cambridge, MA, USA) at a 1:20 dilution overnight at 4°C. Finally, the sections were mounted in AntiFade Mounting Medium with DAPI. All sections were examined under an Olympus microscope (Olympus America Inc. Melville, NY) fitted with epifluorescence imaging and images were captured with a digital camera.

All specimens were sectioned into 5 μm sections from formalin-fixed, paraffin-embedded tissue. Sections were dewaxed in xylene and rehydrated using serial ethanol baths in decreasing concentrations. Specimens were histopathologically evaluated with conventional hematoxylin and eosin (H&E) staining. Further evaluation was performed via immunofluorescence staining. Antigen retrieval was performed with citrate buffer pH 6 (Agilent Dako) in a pressure cooker (BioCare Medical) programmed at 110°C for 15 min. Sections were incubated with primary antibodies rabbit-anti-human CD4 (Novus Biologicals) at 1:200 and mouse-anti-human CD8 (Abcam) at 1:50 overnight at 4°C. Afterward, biotinylated secondary antibodies goat-anti-rabbit (Vector) and horse-anti-mouse (Vector) were both incubated at 1:200 for 1 h at room temperature followed by Streptavidin-AF546 conjugate (Invitrogen) and Streptavidin-AF647 conjugate (Invitrogen), respectively, for 30 min at room temperature. Sections were mounted with ProLong Gold Antifade Reagent with DAPI (Invitrogen) and coverslipped. Immunofluorescence-stained sections were photographed using a fluorescence microscope (EVOS FL Auto 2, Invitrogen) and processed with ImageJ software.

The mouse pancreatic sections were processed as follows: first, they were deparaffinized in xylene, rehydrated in series of alcohol concentrations and double distilled water. Antigen retrieval was then performed using a pressure cooker (Biocare Medical) and 10 mM citrate buffer (pH=6). The sections were blocked with CAS-Block (Life technologies) and incubated overnight at 4°C with the following primary antibodies: guinea pig anti-insulin (1:5; Cat#A0564, DAKO, RRID:AB_10013624), mouse anti-glucagon (1:200; Cat#K79bB10, Abcam, RRID:AB_297642), and rabbit anti-somatostatin (1:200; Cat#ab108456, Abcam, RRID:AB_11158517). DAPI (1:100; Cat#D9542, Sigma-Aldrich) was used for DNA counterstaining. After washing three times in 1xPBS, the sections were incubated with secondary antibodies diluted in 1% PBS/BSA for 2 h at room temperature: guinea pig Cy2 (1:200) for anti-insulin antibody detection, mouse Cy3 (1:300) for anti-glucagon antibody detection, and rabbit Cy5 (1:400) for anti-somatostatin antibody detection. All secondary antibodies were purchased from Jackson ImmunoResearch. Following staining, the sections were mounted with aqueous mounting medium and covered with a coverslip. Images were acquired using an Olympus confocal microscope at 40X magnification. Quantification of images was performed using the ImageJ software.