Cy3 conjugated goat anti rabbit igg

The kidney tissue sections (5 μm) were deparaffinized, rehydrated, and washed with PBS. Cells were fixed in 4% paraformaldehyde, incubated with 0.1% Triton X-100 (Beyotime), and washed with PBS. Tissue sections and cells were blocked in goat serum for 15 min. For TOLLIP-TLR2 and TOLLIP-TLR4 staining, tissue sections were subjected to incubation with anti-TOLLIP (ABclonal, China) and anti-TLR2 (NOVUS, USA)/anti-TLR4 (Santa Cruz, USA) antibodies (1 : 50 dilution) at 4°C overnight, followed by incubation with FITC-conjugated goat anti-rabbit IgG or Cy3-conjugated goat anti-mouse IgG (Beyotime; 1 : 200 dilution) at room temperature for 90 min. For cleaved caspase-3, NLRP3, and p65 staining, tissue sections or cells were subjected to incubation with anti-cleaved caspase-3 (Affinity, China), anti-NLRP3 (ABclonal), and anti-p65 (Affinity) antibodies (1 : 100 dilution) at 4°C overnight, followed by incubation with Cy3-conjugated goat anti-rabbit IgG (1 : 200 dilution) at room temperature for 60 min. Next, tissue sections or cells were subjected to dihydrochloride (DAPI) (Aladdin) nuclear staining. Images were captured using a fluorescence microscope (OLYMPUS).

Immunohistochemistry staining was performed on the paraffin sections of 10-μm thickness. The primary antibodies used for immunohistochemistry were anti-myosin (ZM0196; Zhongshan Golden Bridge, Beijing, China), anti-phospho-Smad1/5/8 (13820S; 1:200, Cell Signaling Technology, Danvers, MA, United States), and anti-Sox9 (ab185966; Abcam, Cambridge, MA, United States). The horseradish peroxidase (HRP)-conjugated anti-rabbit/mouse IgG and the DAB substrate kit purchased from MXB Biotechnologies Inc. (Fujian, China) were used as the secondary antibody and color development reagent, respectively, as the manufacturer instructed. Methyl green or hematoxylin was used for counter-staining.
For Gli1 immunohistofluorescence, the paraffin sections were incubated with the rabbit IgG (514675; ZEn Bio Inc., Chengdu, China) after re-hydration in the gradient ethanol solutions and washing in PBS. The CY3-conjugated goat anti-rabbit IgG (S0011; Affinity, Cincinnati, OH, United States) was applied as the secondary antibody. The Olympus DP72 microscope was used for fluorescence observation and image collection.