Horseradish peroxidase hrp conjugated secondary antibody

Mouse liver samples were fixed in 4% paraformaldehyde and embedded in paraffin for immunohistochemistry. For histopathology, hematoxylin and eosin (H&E) staining of liver tissues was carried out to investigate the liver injury. For immunolocalization, paraffin-embedded mouse liver sections (5 µm) were dried for 1 h at 58 °C, followed by antigen retrieval and incubation with primary antibody (anti-cleaved caspase-3, Cell Signaling Technology, #9661, Danvers, MA, USA, or anti-CD45 antibody, BioLegend, #103107, Amsterdam, Netherlands) in a Ventana automated staining platform (Ventana Medical Systems, Illkirch-Graffenstaden, France). Revelation of primary antibody was carried out using horseradish peroxidase (HRP)-conjugated secondary antibody (Dako, Agilent Technologies, Les Ulis, France) and DAB substrate kit (Ventana Medical Systems, #760-124, Illkirch-Graffenstaden, France). Slides were then counterstained with hematoxylin. All paraffin-embedded liver sections were scanned with a digital slide scanner (Nanozoomer 2.0-RS, Hamamatsu Photonics, Massy, France) and files were analyzed with the NDP viewer 2.5 software (Hamamatsu, Hamamatsu City, Japan). Signal quantifications were performed with an image analysis software (NIS-Element AR analysis software, Nikon, Tokyo, Japan).

Unstained TMA sections of pre-operative curettage tissue were deparaffinized in xylene and rehydrated in a series of ethanol (100%, 95%, 80%, 70% and 50%). Antigen retrieval was performed by boiling for 30 min in an antigen retrieval buffer at pH 9 (Dako, Cat# S2367) before peroxidase blocking (Dako, Cat# S2023) for 8 min. The sections were incubated over night at 4 °C with rabbit anti-human vimentin monoclonal antibody (1:200, D21H3, Fluidigm, Cat# 3143027D, RRID:AB_2811069) followed by incubation with anti-rabbit, horseradish peroxidase (HRP)-conjugated secondary antibody (Agilent Technologies, Cat# K4003) for 30 min. Finally, sections were incubated with diaminobenzidine peroxidase (DAB-chromogen; EnVision detection system, Cat# K3468) for 5 min. Sections were counterstained with Hematoxylin (Dako, Cat# S3301) before hydration and mounting.

Superchip, Inc, was the source of the tissue microarrays of paired samples of adjacent normal tissue and primary CRC tissue, which were used to prepare 4‐μm‐thick paraffin‐embedded sections. All patients diagnosed with CRC had their diagnosis histologically confirmed by biopsy. The tissue sections were processed for deparaffinization/rehydration and antigen retrieval and stained for SIRT2 expression using a primary antibody against human SIRT2 (Cell Signaling Technology). Then, a horseradish peroxidase (HRP)‐conjugated secondary antibody (Dako; Agilent Technologies, Inc) was incubated with the slides. Protein expression was visualized using a 3,3′‐diaminobenzidine chromogenic substrate kit (Dako; Agilent Technologies, Inc). Two independent pathologists blindly scored the immunohistochemistry (IHC) results using a 400× microscope. For IHC of the CRC tissue microarray, each section was scored as a whole. Staining intensity was quantified using the values 3, 2, 1 or 0, indicating strong staining, moderate staining, weak staining or no staining, respectively. The percentage of positive cells was graded as follows: 3, >50%; 2, 10%–50%; 1, < 10%; and 0, negative. The product of staining intensity and grade was calculated and used as the IHC score, which was described as either high expression (>4) or low expression (0‐4) for each sample.

Mouse liver was collected after slaughtering. Liver fragments were fixed in 4% paraformaldehyde and embedded in paraffin. Sections of 4 µm were used for hematoxylin-and-eosin (H&E), Sirius Red and immunohistochemistry stainings. For immunolocalization of glutamine synthetase (GS, Abcam, ab73593, 1/100), or of CD45 (BioLegend, #103107, 1/30), tissue sections were dried 1 h at 58 °C, followed by antigen retrieval and incubated with the corresponding primary antibody in a Ventana automated staining platform (Ventana Medical Systems, Illkirch-Graffenstaden, France). Revelation of primary antibody was carried out using horseradish peroxidase (HRP)-conjugated secondary antibody (Dako, Agilent Technologies, Les Ulis, France) and DAB substrate kit (Ventana, #760-124). Slides were then counterstained with hematoxylin.
All paraffin-embedded liver sections were scanned with a digital slide scanner (Nanozoomer 2.0-RS, Hamamatsu Photonics, Massy, France) and files were analyzed with the NDP viewer 2.5 software (Hamamatsu). Quantification of Sirius Red, CD45 or glutamine synthetase positive signals was performed with an image analysis software (NIS-Element AR analysis software, Nikon, Tokyo, Japan).

A total of 20 μg of whole cell lysate were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane (Merck Millipore, Darmstadt, Germany), as previously described [37 (link)]. Membranes were immunoblotted with anti-human FBXW7 monoclonal antibody (1:1000 dilution, clone SP237, Abcam) followed by horseradish peroxidase (HRP)-conjugated secondary antibody (1:4000 dilution, Dako). HRP-conjugated ACTB antibody (1:8000 dilution, clone AC-15, Sigma-Aldrich, St. Louis, MO, USA) was used as a loading control.