Horseradish peroxidase conjugated anti rabbit secondary antibody

The livers of mice were excised for each group, fixed in 4% formalin, and embedded in paraffin. Sectioned tissues were stained with haematoxylin and eosin (H&E) and Sirius Red. The hepatic fibrosis grade was analysed using Sirius-Red-stained tissues. Immunohistochemical staining was performed by incubating the tissue sections with a primary antibody against αSMA (dilution 1:500, Abcam, Cambridge, UK) in a moisturised chamber at 4 °C overnight. After that, they were incubated with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (Dako, Glostrup, Denmark).

Equal amounts of protein were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a 0.45-µm polyvinylidene difluoride (PVDF) immunoblot membrane (Amersham, Amersham, UK). After membrane blocking with 5% skimmed milk in PBS with 0.01% Tween 20 (wash buffer) for 1 h at room temperature (RT), the membranes were incubated overnight at 4°C with commercial primary antibodies against phospho-AKT (Cell Signaling, Danvers, MA, USA), HO-1 (Santa Cruz Biotechnology, Dallas, TX, USA), NRF2 (Santa Cruz Biotechnology), histone (Cell Signaling), β-actin (Sigma-Aldrich, St Louis, MO, USA), and PARP (Cell Signaling). The next day, the membranes were washed three times and then incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:2,000; Dako, Carpinteria, CA, USA) for another 1 h at RT. Blots were visualized with a densitometric ECL kit (Amersham) and analysis was performed using ImageJ software.
To analyze the effects of kinase inhibitors on the expressions of Nrf2 proteins, seeded raw cells in dishes were pretreated with wortmannin (200 mM) for 3 h; then, the cells were washed and infected with the Leishmania promastigotes for different times. The cell lysates prepared using the lysis buffer, as described above, were subjected to Western blot analysis.

Dot blot apparatus was assembled with nitrocellulose membrane and filter paper, following instructions of the manufacturer (Bio-Rad). SMC conditioned medium supernatant (100 μl) was applied to each Dot blot well, and the nitrocellulose membrane was allowed to dry for 1 h. The membrane was then blocked in 5% dry milk in tris-buffered saline (TBS) containing 0.1% Tween-20, and incubated in rabbit anti-CCL2 antibody (1:1000, Abcam) overnight at 4 °C. The next day, after washing in TBS with 0.1% Tween-20, the membrane was incubated with horseradish peroxidase–conjugated anti-rabbit secondary antibody (1:2000; DAKO) for 1 h. Detection was performed with the Western Blotting Substrate Plus (Pierce) and GBOX imaging system (Syngene).

Scaffolds were fixed and embedded in paraffin in two orientations and cut into 5 µm sections. Cell morphology and distribution were assessed using Haematoxylin and Eosin staining and RGB staining [43 (link)]. For RGB staining, sections were stained with 1% Alcian blue in 3% aqueous acetic acid (pH 2.5) for 20 min and rinsed in tap water. Following this, sections were stained with 0.04% fast green in distilled water for 20 min and rinsed in tap water for 5 minutes. Lastly, sections were stained with 0.1% Picrosirius red for 30 min, followed by 2 changes of 5 min in 1% acidic acid in tap water. For type I and II collagen immunohistochemistry, antigen were retrieved using 1 mg/mL pronase (Sigma-Aldrich) followed by blocking with 5% bovine serum albumin (BSA) in PBS for 30 min. Samples were incubated with the primary antibody (type I collagen, rabbit monoclonal 1/400 in PBS/BSA 5% or type II collagen, mouse monoclonal 1/100 in PBS/BSA 5%) overnight. Sections were incubated with horseradish peroxidase conjugated anti-rabbit secondary antibody (DAKO, Glostrup, Denmark) for 30 min after washing. Immunoreactivity was visualized using diaminobenzidine peroxidase substrate solution (DAB, Sigma-Aldrich) and sections were counterstained with Mayer’s hematoxylin.

PAGE gels were electrotransferred onto nitrocellulose or PVDF membranes (Ambion). The membranes were blocked with 5% milk in PBS-T (PBS with 0.1% v/v Tween) and incubated overnight at 4 °C with primary antibody (Table 2) in PBS containing 1% bovine serum albumin (BSA) and 2 mM NaN₃. After extensive washing in PBS-T, the blots were incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (Dako), washed again in PBS-T, and developed with ECL before imaging (ImageQuant LAS4000, GE Healthcare, Chicago, IL, USA). The CP from one mouse only yields in the range 60–100 µg of protein sample. To increase the number of blots per experimental animal, the membranes were divided for high-, medium-, and low-molecular proteins prior to antibody incubation. For dual immunoblotting native PAGE, secondary antibodies were coupled to either 700 nm or 800 nm IR Dye and imaged on a LiCor Odyssey Imager (LiCor Biosciences, Lincoln, NE, USA).

The TMAs were deparaffinized and rehydrated by using xylene and a graded series of ethanol. Antigen repair was performed in citric acid buffer (PH6.0). Then, the slices were incubated at room temperature in 3% (v/v) hydrogen peroxide solution to block the activity of endoperoxidase. Nonspecific sites were blocked up with 3% bovine serum albumin (BSA), and incubated with a primary rabbit monoclonal anti-TRIM24 antibody (1:200 dilution, ab174287, Abcam, England). The sections were then incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (Dako, America). Colors were presented with a DAB kit (Dako, America). Then, nucleus was counterstained with hematoxylin, dehydration and mounting. TBS containing 1% BSA was used as a substitute for the primary antibody as a negative control.
TRIM24 immunohistochemical staining was scored according to intensity and percentage of TRIM24-positive cells by two experienced pathologists independently. Staining intensity was graded from 0 to 2(0, negative; 1, weak; 2, strong). The percentage of TRIM24-positive cells was also scored from 1 to 4 (0–10%, 11–25%, 26–75%, 76–100%). A score ranging from 0 to 8 was calculated by multiplying the staining intensity score with the staining percentage of TRIM24-positive cells score, resulting in a low (0–3) level or a high (4–8) level for each sample.