Ihc tek epitope retrieval solution

Primary antibodies used are summarized in the Table. Immunostaining was performed as reported previously by our group [16 (link)–18 (link)]. Briefly, serial coronal sections were mounted on 3‐aminopropyl triethoxysilane‐coated slides. Every eighth section from the habenular to the posterior commissure (8–10 sections per animal) was examined using unbiased stereological principles. The sections for testing Aβ (4G8 antibody) were deparaffinized, hydrated, and pretreated with formic acid (88%) and subsequently with 3% H₂O₂ in methanol. The sections for testing total tau (HT7 antibody), and phospho‐tau epitopes, were deparaffinized, hydrated, subsequently pretreated with 3% H₂O₂ in methanol, and then treated with citrate (10mM) or IHC‐Tek Epitope Retrieval Solution (IHC World, Woodstock, MD) for antigen retrieval. Sections were blocked in 2% fetal bovine serum before incubation with primary antibody overnight at 4°C. Next, sections were incubated with biotinylated anti‐mouse immunoglobulin G (Vector Laboratories, Burlingame, CA) and then developed by using the avidin‐biotin complex method (Vector Laboratories) with 3,3′‐diaminobenzidine as a chromogen. Light microscopic images were used to calculate the area occupied by the immunoreactivities by using the software Image‐Pro Plus for Windows version 5.0 (Media Cybernetics, Bethesda, MD).

Paraffin-embedded femur and tibia samples were sectioned at 10 μm and subjected to immunostaining for PCNA. Antigen retrieval was performed on the sections with IHC-Tek Epitope Retrieval solution (IHC World, Woodstock, MD, USA) at 70°C for 20 minutes. Sections were blocked in 1% Donkey IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) with 0.2% ovalbumin (Sigma-Aldrich). Rabbit anti-PCNA antibody (1:400; Cell Signaling Technology, Danvers, MA, USA) was applied over-night at 4°C, followed by biotinylated anti-Rabbit IgG (Abcam, Cambridge, UK) and peroxidase-conjugated streptavidin (Jackson ImmunoResearch Laboratories) incubation, and visualized by diaminobenzidine substrate (DAB; Life Technologies, Carlsbad, CA, USA). PCNA-positive cells were counted using ImageJ software (US National Institutes of Health, Bethesda, MD, USA).

FXR1 and cMYC protein levels in human ovarian cancer tissues were analyzed using three TMAs (Cat# OV1005bt, Cat# OVC961 and Cat#OV1004, US Biomax Inc., Rockville, MD). For this purpose, the slides were dewaxed in xylene, and rehydrated through graded ethanol to distilled water. Antigen retrieval for the slide specimens were performed using IHC-Tek epitope retrieval solution and steamer set (IHC World, LLC.). The slides were then immersed in 3% H₂O₂ for 10 min to quench endogenous peroxidase followed by blocking with 10% goat serum for 1 h. Vectastain ABC-AP Kit (Vector Labs, Burlingame, CA) and Vector Red Alkaline Phosphatase Substrate Kit I (Vector Labs, Burlingame, CA) were used for tissue staining as per manufacture protocol. FXR1 primary antibody (Proteintech, Cat#13194-1-AP) was used at 1:200 dilution and cMYC primary antibody (Santacruz Biotechnology, Cat#sc-47694) at 1:100. Following, Vector red staining, the slides were counterstained with Harris modified hematoxylin (Thermo Fisher Scientific Inc., Rockford, IL), dehydrated with graded ethanol and xylene, and finally mounted with paramount. TMAs slides was digitally scanned using Panoramic 250 FLASH III scanner (3D HISTECH ltd. Version 2.0) and, using the Case Viewer software (3D HISTECH ltd. Version 2.0) was used to view and analyze images.

Tumor tissues were harvested and fixed for 48 – 72 hours in 4% paraformaldehyde (4%PFA, Boston BioProducts) and stored in 70% ethanol until further processing. Paraffin-embedded tumor sections (10 μm) were deparaffinized followed by heat-mediated antigen retrieval for 30 minutes in IHC-TEK epitope retrieval solution (IHC World). Following antigen retrieval, tumor sections were permeabilized with 100% methanol. Sections were blocked for 30 minutes with Tris-NaCl (TNB) blocking buffer (PerkinElmer), and then incubated with rabbit anti-vaccinia virus antibody (Abcam) diluted 1:100 in TNB blocking buffer overnight in a humidified chamber at 4°C. Following incubation, tumor sections were wash and incubated with Alexa-Fluor-488-conjgated goat anti-rabbit secondary antibody (Abcam) for 1 hour at room temperature. Finally, the sections were counterstained with DAPI.
Immunohistochemistry for CD3 and CD8 were performed by the Pathology Core at City of Hope. Briefly, deparaffinized tumor sections (10 μm) were stained with hematoxylin & eosin (H&E, Sigma-Aldrich), rat anti-mouse CD3 (Abcam), and rat anti-mouse CD8a (Novus Biologicals). Images were obtained using the Nanozoomer 2.0HT digital slide scanner and the associated NDP.view2 software (Hamamatzu). For CD8 quantification after IHC staining, ImageJ (NIH) analysis was performed as per the standard recommended algorithm^{55 (link)}.