Goat anti rabbit igg h l

For α-SMA staining, the following unstained slides were deparaffinized through standard methods. 3% H₂O₂ was used to inactivate any endogenous peroxidase for 30 min. The lung slices were blocked in Tris-buffered saline (TBS) with 5% bovine serum albumin (BSA, Solarbio, China) for 1 h. Then, slides were incubated with α-SMA antibody (1: 200, SAB) at 4°C overnight. Slides were washed with 1× TBS and then incubated with Goat Anti-Rabbit IgG H&L (1: 2000; Abcam) for 30 min. The nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI, Invitrogen, USA). After washing the slides with PBS 3 times, slides were then mounted with 90% glycerol and detected by a fluorescence microscope (Nikon, Japan).
For PPARγ staining, the samples were fixed with PFA and blocked with 5% BSA supplemented with 0.4% Triton-X for 1 h at room temperature. Fixed samples were incubated with the primary PPARγ antibody (Sigma-Aldrich, 1 : 200) at 4°C overnight. After washing, the samples were incubated with the Goat Anti-Rabbit IgG H&L (1: 2000; Abcam) for 30 min and counterstained with DAPI. After mounting, the samples were detected by a fluorescence microscope (Nikon TS2R, emission spectrum 488 nm, absorption spectrum 519 nm).

The following primary antibodies were used in this study: anti-CD59 [MEM-43] (cat. no. ab9182; Abcam, Cambridge, UK), anti-cyclin D1/FSTL3 (cat. no. bs-0623R; Bioss), anti-osteopontin (cat. no. ab33046; Abcam), anti-NF-κB p105/p50 [E381] (cat. no. ab32360; Abcam), anti-RANK [EPR4740(N)] (cat. no. ab182158; Abcam), anti-RANKL [12A668] (cat. no. ab45039.7; Abcam), and anti-RANK (cat. no. ab222215; Abcam).
The following secondary antibodies were used in this study: goat anti-mouse IgG H&L (Alexa Fluor 488; cat. no. ab150113; Abcam), goat anti-rabbit IgG H&L (Alexa Fluor 488; cat. no. ab150077; Abcam), goat anti-rabbit IgG H&L (horseradish peroxidase (HRP)) preadsorbed (cat. no. ab97080; Abcam), and goat anti-mouse IgG H&L (HRP) preadsorbed (cat. no. ab97040; Abcam).

The procedure of IHC assay was in accord with a previous study.¹⁹ In brief, the goat polyclonal anti‐TRPC1 antibody (1:150; Abcam) was applied as primary antibody, and the rabbit anti‐goat IgG (H&L) (1:2000; Abcam) was applied as secondary antibody. The experiment was carried out in strict accordance with the instructions. After IHC assay, the expression of TRPC1 protein was assessed by semi‐quantitatively method based on the intensity and density of positive carcinoma cells. IHC staining intensity was classified as four grades: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). IHC staining density was classified as five grades: 0 (0%), 1 (1%‐25%), 2 (26%‐50%), 3 (51%‐75%), and 4 (76%‐100%). The max IHC score was 12 which was a product of two above staining scores.

TRPC1 protein expression was examined by immunohistochemistry (IHC), and the experimentation of the IHC assay was in accord with a previous study.¹⁷ Briefly, the collected FFPE specimens were cut into slides. The slides were depolymerized in xylene, rehydrated, and then heated in a citric acid buffer at pH 6 to expose antigens. Sequentially. The slides were incubated with goat anti‐TRPC1 antibody (dilution 1:150; Abcam) as the primary antibody and rabbit anti‐goat IgG (H&L) (dilution 1:2000; Abcam) as the second antibody. After incubation, diaminobenzidine and hematoxylin were used for staining. The results of the IHC assay were semi‐quantitatively evaluated using a light microscope by 2 investigators who were blinded to the patient's clinical data according to the density and intensity of stained cells. The density was scored as 5 grades (score 0–4), and the intensity of stained cells was scored as 4 grades (score 0–3). The final score of the IHC assay was 12, which was a product of the density score and the intensity score. The final score of the IHC assay was a product of the density score and the intensity score, ranging from 0 to 12.