Affinipure goat anti rabbit igg h l

Whole-cell extracts were extracted by directly lysing the cells with 1× radioimmunoprecipitation assay buffer (Beyotime) with 1 mM PMSF (Beyotime) added immediately before use. Samples were boiled by adding 6× SDS sample buffer for 10 min at 100 °C and resolved using SDS-polyacrylamide gel electrophoresis. The proteins were probed with the following antibodies: monoclonal anti-GFP (1:2000 dilution; Thermo Fisher Scientific), anti-βACTIN (1:2000 dilution; Thermo Fisher Scientific), and anti-METTL3 (1:1000 dilution; Abcam), and anti-FTO (1:2000 dilution; Abcam). Immunodetection was performed using horseradish peroxidase–conjugated Affinipure goat anti-mouse IgG (H + L) (1:5000 dilution; catalog no.: SA0 001-1; Proteintech) or horseradish peroxidase–conjugated Affinipure goat anti-Rabbit IgG (H + L) (1:5000 dilution; catalog no.: SA00001-2; Proteintech) and ECL prime substrate (Bio-Rad) according to the manufacturer's instructions.

For western‐blotting, HeLa cells were plated in 12‐well plates and transfected with 1 µg plasmid. After 48 h transfection, cells were washed with PBS and lysed in 50 µL 1 × SDS loading buffer (50 mm Tris‐HCl pH 6.8, 10% glycerol, 2% SDS, 0.1% bromophenol blue, 1% beta‐mercaptoethanol) at room temperature for 10 min. The cell lysate was collected and then boiled at 95 °C for 10 min. The appropriate amount of protein was loaded onto SDS PAGE gels. The separated proteins were transferred onto a PVDF membrane (Millipore) in an ice‐bath for 2 h. Then the PVDF membrane was blocked in 5% (w/v) BSA (Beijing Dingguo changsheng Biotechnology Co.,Ltd, FA016) in TBST (Tris‐buffered saline, 0.1% Tween 20) at room temperature for 1 h. The blot of protein was stained as indicate for at least 12 h at 4 °C. The blot was washed four times with TBST at room temperature for 5 min each, then stained with 1:5000 HRP‐conjugated Affinipure Goat Anti‐Rabbit IgG(H+L) (Proteintech, SA00001‐2) or HRP‐conjugated Affinipure Goat Anti‐Mouse IgG(H+L) (Proteintech, SA00001‐1) in 5% BSA (w/v) in TBST for 1 h at room temperature. The blots were washed four times with TBST at room temperature for 5 min each time and imaged on Molecular Imager ChemiDocTM XRS+ Imaging System (Bio‐Rad) after incubation with Rhea ECL (US Everbright, Inc).

Cells were lysed by scraping in 2X sodium dodecyl sulfate (SDS) buffer (100 mM Tris-HCl, pH 6.8, 20% glycerol, 4% SDS) with 1× Complete Mini EDTA-Free Protease Inhibitor Cocktail (Roche, 11697498001) and boiling for 5 min. Total protein content of cell extracts was determined using a Bicinchoninic Acid (BCA) Kit (ThermoFisher Scientific, 23227). Total protein lysates (15 μg) were run on Mini-Protean TGX 4–20% gradient gels (Bio-Rad, 456–1093) and transferred onto InvitrolonTM PVDF membranes (Invitrogen, LC2005). Transfer was visualized with Ponceau staining and the membrane was blocked with 5% nonfat dry milk in TBST (20 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween 20 [P1379, Sigma-Aldrich]). The membrane was incubated with primary antibody overnight at 4°C followed by 1 h incubation at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibody; BD Pharmingen HRP Goat Anti-Mouse Ig (BD Biosciences, 554002) or HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (Proteintech, SA00001–2). Signal detection was performed with a western blotting chemiluminescent reagent (Sigma-Aldrich, CPS3500) and an iBright Imaging System (ThermoFisher Scientific).

Monolayer cells were cultured on glass coverslips until they attained 70–80% confluence and were then fixed with 75% ethanol for 30 min. After being washed with PBS thrice, these slides were incubated with endogenous peroxidase inhibitor for 15 min, and blocked with goat serum for 20 min, followed by treatment with different primary antibodies at 4°C overnight (information regarding the antibodies used has been listed in Table S1). The slides were then incubated with biotin-labeled goat anti-mouse/rabbit IgG for 20 min and horseradish-labeled streptomycin for 15 min. Detailed ICC analysis was performed referring to the instructions of Biotin-Streptavidin HRP Detection Systems (SP-9000, Zhongshan Inc.). PBS was used as a negative control to replace the primary antibody. For immunofluorescence staining, the cells were incubated with primary antibodies 4°C overnight (information regarding the antibodies used has been listed in Table S1), followed by reacting with the fluorescein (FITC)-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) (SA00003-2, Proteintech) at room temperature for 30 min. The nuclei were visualized with DAPI. The cell morphology was visualized and photographed with fluorescence microscopy.

Haematoxylin–eosin (HE) and immunohistochemistry (IHC) staining were performed as described in our previous study.⁴¹ In brief, tissues were paraffin‐embedded, dewaxed, rehydrated and stained with HE and Masson. For IHC, tissue sections of lungs from mice were incubated with antibody against mouse α‐SMA (19245S, CST, USA), followed by incubation with biotin‐conjugated Affinipure Goat Anti‐Rabbit IgG (H + L) (SA00004‐2, Proteintech, China). Further, the images were captured using a Digital Pathology Scanner (Aperio CS2, Leica). The mean linear intercept (MLI) was then used to estimate the average diameter of a single alveolus using the formula:
MLI = Total length/Alveolar septal number.
Airway wall thickness was assessed by segmental airway wall area percentage (Segmental WA%  =  [Outer bronchus area − Airway luminal area]/Outer bronchus area).
ImageJ software was used to measure airway area and Masson positive area and to calculate as percentage of stained area to total airway wall area (collagen area/airway area). The same statistical methods were used for IHC of α‐SMA (α‐SMA area/airway area) and FN1 (FN1 area/airway area).

The cells were placed in cold RIPA solution (Solarbio, C0065) with PMSF (Solarbio, P0100) after washing the cells twice with phosphate buffer. Subsequently, they were pyrolysed with a protease inhibitor mixture for 30 min. The supernatant was centrifuged and harvested to analyse the protein concentration using the BCA Protein Assay Kit (BestBio, A045-4). Immunoblotting was conducted using the SDS-PAGE electrophoresis system. Briefly, 25 μL of sample was loaded and electrophoresed on a 10% SDS reducing gel. Blots were blotted onto a polyvinylidene fluoride membrane and incubated with primary antibodies against TLR3 (1 : 1000, Abcam, ab13915, USA), TLR4 (1 : 500, Abcam, ab13556), TLR6 (1 : 1000, Proteintech, 22240-1-AP), IDO1 (1 : 500, Abcam, ab55305), and NF-κB (1 : 500, Abcam, ab14059) overnight at 4°C. On the following day, the blot was washed three times with TBST and incubated with horseradish peroxidase-conjugated (HRP-conjugated) AffiniPure Goat anti-Mouse IgG (H+L) (1 : 5000, Proteintech, SA00001-1, USA) or HRP-conjugated AffiniPure Goat anti-Rabbit IgG (H+L) (1 : 5000, Proteintech, SA00001-2). Further, the blot was kept at 25°C for 1 h in TBST. Finally, proteins on the washed membrane were visualised using the TBST (Solarbio, T1082).