3 3 diaminobenzidine substrate chromogen system

Paraffin-embedded liver tissue wax blocks were sectioned at 5 μm. After being dewaxed and rehydrated, the sections were incubated in 3% hydrogen peroxide/methanol. Heat-induced antigen retrieval was performed by heating in 10 mM sodium citrate buffer for 10 min. Sections were incubated in anti-Beclin1 antibody (Cell Signaling Technology) at 1:200 dilution at 4 °C overnight. 3,3′-Diaminobenzidine Substrate Chromogen System (Dako, Carpinteria, CA, USA) was employed during the detection procedure. Subsequently, the sections were counterstained using hematoxylin for 10 s. Finally, after being dehydrated in ethanol, cleared in xylene and mounted, the sections were observed in the light microscope by a pathologist who was initially blinded to treatment groups, and five random fields of each slide were semi-quantified and averaged using the software ImageJ 1.48 (National Institutes of Health, Bethesda, MD, USA) according to its instructions, then come up with the data of density of target protein positive cell, and the relative density (/sham) of protein represents the protein expression level of HO-1.

Lungs were fixed in 10% phosphate-buffered saline (PBS)–formalin for at least 24 h and embedded in paraffin. Immunohistochemical analysis of paraffin-embedded sections (4 μm) was performed using Vectastain Universal Quick Kit (Vector Laboratories Inc., Burlin, CA, USA) according to the manufacturer’s recommendations. Briefly, slides were washed in gradual dilutions of ethanol for deparaffinization. Antigen retrieval was achieved by incubating the slides in heated (100 °C) citrate buffer (pH = 6.0; Dako, Santa Clara, CA, USA). Endogenous peroxidase activity was suppressed by 3% hydrogen peroxide treatment. Then, slides were incubated with a blocking buffer (1% universal horse serum) for 1 h at room temperature, the primary antibody overnight at 4 °C, and with an HRP-labeled secondary antibody for 30 min at room temperature. Positive staining was detected using a 3,3′-diaminobenzidine substrate chromogen system (Dako), which was followed by counterstaining with hematoxylin. Primary antibodies used were CD68 (1:100, BioRad, Hercules, CA, USA), CD163 (1:100, Abcam, Cambridge, MA, USA), and acetyl-tubulin (1:100, Santa Cruz Biotechnology, Dallas, TX, USA).

IHC was conducted as described previously [40 (link)]. Brain samples were embedded in paraffin, and tissue sections were prepared using HistoCore AutoCut (Leica, Deerfield, IL, USA). Next, the sections were treated with 3% hydrogen peroxide/methanol and with 0.25% pepsin to retrieve antigens. The samples were incubated in blocking solution (Dako, Carpinteria, CA, USA), and were incubated at 4 °C overnight with the primary antibodies diluted in the antibody diluent (Dako). Then, the sections were washed with Tris-buffered saline with 0.1% Tween 20 and incubated with a HRP conjugated secondary antibody (Dako). A 3,3′-diaminobenzidine substrate chromogen system (Dako) was used to detect antibody binding. Stained sections were visualized with an Olympus IX71 inverted microscope (Olympus Optical, Tokyo, Japan). The IHC images were quantified by transforming images into positive/negative pixels using ImageJ and validated by statistical analyses.