Expose mouse and rabbit specific hrp dab detection ihc kit

After sacrificing, the hearts were extracted, fixated in 10% formaldehyde, and immersed in paraffin. Formalin-fixed paraffin-embedded (FFPE) sections, 5 μm thick, were deparaffinized, rehydrated, and treated with citrate buffer (pH 6.0) in a microwave for antigen restoration. Endogenous peroxidase activity was blocked using hydrogen peroxide (3%). Immunohistochemical staining was performed by incubating FFPE tissue sections with primary mouse anti-cytochrome C antibody (338500, Invitrogen, Carlsbad, CA) overnight at room temperature. Staining was visualized by using Expose Mouse and Rabbit Specific HRP/DAB Detection IHC Kit (ab80436, Abcam, Cambridge, UK). Sections were counterstained with Mayer's hematoxylin and photomicrographed by light microscope (Olympus BX51, Japan) equipped with a digital camera. Results are presented as a mean count of positive stained cells per field at ×40 magnification [19 (link)].

Rats were anesthetized and sacrificed by perfusing through the heart with 4% paraformaldehyde in PBS (pH 7.4). Brains were removed, postfixed overnight and sectioned by the Department of Pathology, Tianjin Huanhu Hospital. The sections were pretreated with xylene and then incubated with primary antibody against rat Fas ligand (Abcam Anti-Fas Ligand antibody, ab15285), at 4 °C overnight. Next, sections were incubated with complement, HRP conjugate, and DAB, orderly (Abcam EXPOSE Mouse and Rabbit Specific HRP/DAB Detection IHC kit, ab80436). Next, sections were counterstained with hematoxylin. Images were captured by a microscopy (Olympus).
The total FasL immunostaining score was computed according to Wang Y et al.43 (link). Briefly, the percentage of positive was defined as 0 (<5%, negative), 1 (5–25%, sporadic), 2 (25–50%, focal), or 3 (>50%, diffuse). Staining intensity was defined as 0 (no staining), 1 (weak staining), 2 (moderate staining), or 3 (strong staining). The total score of immunostaining was computed as the percentage positive score × staining intensity score, and ranged from 0 to 9. We examined five successive fields as a section and summed the scores.

For immunohistochemistry (IHC), tissue microarray (TMA) slides of the TMU-SHH HCC cohort were established, then heat-based antigen retrieval was performed in EDTA-containing buffer, sections blocked with 5% bovine serum albumin (BSA)/1% HISS/0.1% Tween20 solution and incubated with primary recombinant antibody against PDK1 (1:400 dilution; Anti-PDK1 antibody, ab90444) overnight, at 4 °C. PDK1 immunoreactivity/positivity was detected using the mouse IgGk light chain binding protein conjugated to horseradish peroxidase m-IgG BP-HRP (#sc-516102; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and the EXPOSE mouse and rabbit specific HRP/DAB detection IHC kit (#ab80436, Abcam plc., Cambridge, MA, USA). This study was approved by the Institutional Human Research Ethics Review Board (TMU-JIRB No. 201302016) of Taipei Medical University.

The avidin biotin-peroxidase complex method was used for immunostaining of formalin-fixed, paraffin-embedded sections. Sections were deparaffinized and rehydrated. The antigen was retrieved using boiling sodium citrate. Sections were incubated overnight at room temperature with rabbit polyclonal primary antibody anti-UCP1, anti-TBXI, anti-TMEM26 and anti-Cytochrome C, and for different adipocyte superficial markers including amino acid transporter asc-1 (ASC-1), purinergic receptor P2X (P2RX5), proton-coupled and amino acid transporter 2 (PAT2); eosinophil markers CD137, CD38 and Anti-Sicglec8 (Abcam, United Kingdom). The Expose Mouse and Rabbit Specific HRP/DAB Detection IHC Kit was used for detection following manufacture instructions (Abcam, United Kingdom). Staining was achieved adding diaminobenzidine substrate (DAB). Immunohistochemical stains were digitally analyzed by quantitative computerized image software using the Aperio CS scanner (San Diego, CA, United States).

All human tissues were obtained from surgical resection specimens of HCC patients at Taipei Medical University-Shuang-Ho Hospital (New Taipei City, Taiwan). Tissue microarray (TMA) slides were established, then heat-based antigen retrieval was performed in EDTA-containing buffer, sections blocked with 5% bovine serum albumin (BSA)/1% HISS/0.1% Tween20 solution, and incubated with primary recombinant antibody against MALAT1 (1:400 dilution; #MOB-4044z, Creative Biolabs, NY, USA) overnight, at 4 °C. MALAT1 immunoreactivity/positivity was detected using the mouse IgGκ light chain binding protein conjugated to horseradish peroxidase m-IgGβ BP-HRP (#sc-516102; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and the EXPOSE mouse and rabbit specific HRP/DAB detection IHC kit (#ab80436, Abcam plc., Cambridge, MA, USA). This study was approved by the Institutional Human Research Ethics Review Board (TMU-JIRB No. 201302016) of Taipei Medical University.

The sections were pretreated with xylene and then incubated with primary antibody against rat proliferating cell nuclear antigen (PCNA) or Caspas-3 (Santa Cruz Biotechnology, Inc.), at 4 °C overnight. Next, sections were incubated with complement, horseradish peroxidase (HRP) conjugate, and diazoaminobenzene (DAB), orderly (Abcam EXPOSE Mouse and Rabbit Specific HRP/DAB Detection IHC kit). Next, sections were counterstained with hematoxylin. Images were captured by a microscopy (Olympus). The images of immunostained sections were analyzed using Image Pro-Plus (IPP) software (Media Cybernetics, Silver Spring, MD, USA) to calculate the density mean, area sum, and integrated optical density (IOD) of positive expression. The PCNA and Caspase-3 levels were denoted by Average optical density (AOD). AOD = IOD(sum)/Area(sum).

Immunohistochemical staining, for IL-6, CCL21 and for CD4+ and CD8+ cells, was performed in dewaxed paraffin sections. Sections were dewaxed in Histochoice (VWR, Visalia, CA, USA), passed through graded alcohols, and rehydrated with water. Then, antigen retrieval was performed by incubating sections in bowled citrate buffer for 15 min at room temperature. After blocking with Protein Block (Abcam kit, Burlingame, CA, USA), sections were incubated overnight with rabbit anti-rat polyclonal IL-6, CD4-antibody (Novus Biological, CO, USA), CCL21-antibody (Invitrogen/Thermo Fisher, Scientific, Waltham, MA, USA) or mouse anti-rat monoclonal CD8 antibody (Thermo Fisher) at 4°C. Then, the sections were treated with hydrogen peroxide blocking reagent at room temperature and were processed using EXPOSE Mouse and Rabbit specific HRP/DAB Detection IHC Kit (Abcam kit, cat#80436) according to the manufacturers’ instructions, followed by counterstaining with Mayer’s hematoxylin solution (IHC World LLC, Ellicott city, MD, USA). As negative control, 2.5% normal goat serum was used to confirm specific staining of secondary antibodies. Stained sections were examined by light microscopy. Data was quantitated using ImagePro.Plus software. For quantification, 3 sections at 100 μm interval were analyzed from each knee, and for CD4 and CD8, we focused on synovial tissue at the joint capsule.