Startingblock t20 blocking buffer

Paraffin-section containing aortic tissue and PVAT were deparaffinized, dehydrated, and boiled for epitope retrieval using an antigen retrieval buffer at pH = 6.0 (Opal 4-color IHC Kit, PerkinElmer). Tissue sections were blocked (StartingBlock™ T20 Blocking Buffer, 37539; Thermo-Fisher Scientific) for 1 h and incubated with primary antibodies (cathepsin S: ab18822, Abcam; MMP-12: PA5-13181, Thermo-Fisher Scientific; F4/80: ab6640, Abcam; CISD1: 16,006–1-AP, Proteintech; and NF-κB p65: SC-372, Santa Cruz) overnight at 4℃. The sections were incubated with HRP-conjugated secondary antibodies for 1 h and administrated with 50 × diluted Opal working solution for 10 min at room temperature. The following primary antibodies were stained with a repeat procedure including antigen stripping and blocking steps. DAPI was used to stain cell nuclei via adding mounting media containing fluoroshield with DAPI. The images were visualized by confocal microscopy (C1-Si, Nikon).

Cortical brain tissue samples were harvested, and protein and RNA extraction were done using mirVana PARIS Kit (Ambion) according to the manufacturer's instructions. Protein content was determined by the BCA protein assay kit (Novagen) using bovine serum albumin (BSA) as a standard. 20 µg protein lysate was mixed with 4x Sample Loading Buffer (Biorad) and 20x Reducing Agent (Biorad) heated for 5 minutes at 95°C and separated by SDS-PAGE in 12% Criterion XT Bis-Tris precast gels (Biorad). After transfer (1 hr, 40V) to a nitrocellulose membrane (Whatman), membranes were blocked (StartingBlock T20 blocking buffer, Thermo Scientific) for 15 minutes at room temperature and incubated overnight at 4°C with 0.2 µg/ml polyclonal rabbit anti-myelin-basic-protein antibody (Genscript). Membranes were rinsed again with 0.1% TBST then incubated with 0.2 µg/ml horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Santa Cruz Biotech) for 1 hr at room temperature. Signals were detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and quantified using Bio-Rad2000 gel imaging system with QUANTITY ONE software (Biorad). Monoclonal mouse anti-beta actin antibody conjugated to HRP was used as a loading control at a concentration of 31 ng/ml.

Following in vitro assays, tumor cells were disrupted in lysis buffer (CST) on ice for 10 min. Lysates were clarified by centrifugation and equal amounts of proteins resolved by SDS-PAGE before transfer to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked with Starting Block T20 blocking buffer (Thermo Scientific, Cambridge, MA, USA) and immunoblotted with the indicated antibodies. Antibody-antigen complexes were visualized using an Odyssey system (LI-COR, Lincoln, NE, USA).

For the indirect ELISA, a 96-well plate was coated with 2 μg/ml of the S1, RBD, and N proteins (Sino Biological, China) diluted in phosphate-buffered saline (PBS) overnight at 25°C. Each well was blocked with 300 μL of StartingBlock T20 blocking buffer (Thermo Fischer Scientific, Waltham, MA, USA) for 1 h at 25°C. Heat-inactivated convalescent-phase serum samples from COVID-19 patients and healthy donors were diluted in blocking buffer at a ratio of 1:200, and then 100 μL of the samples was added to the 96-well plate in duplicate. After the plates were washed, horseradish peroxidase (HRP)-tagged anti-human (IgG, IgM, and IgA) antibodies (Abcam, Cambridge, UK) were diluted 1:10,000 with blocking buffer and added to the wells (100 μL/well), and the plate was then incubated for 1 h at 25°C. Samples with N antibodies were incubated only for 30 min at 25°C because of the higher signal. The chromogenic reagent 3,3′,5,5′-tetramethylbenzidine (TMB) was mixed with an equal volume of color A and color B (R&D Systems, Minneapolis, MN, USA). The TMB reaction time for the S1 and RBD ELISA was 5 min, whereas that for the N protein ELISA was 10 min. After the reaction, stop solution (R&D Systems) was added to the wells and the OD was measured immediately at 450 nm using a Synergy 2 microplate reader (Bio-Tek, Winooski, VT, USA).

Proteins were extracted from the proximal colon tissues and analyzed on Ready Gel Tris-HCl (Bio-Rad). The tissues were homogenized in RIPA buffer (1% IGEPAL, 0.5% sodium deoxycholate, and 0.1% SDS in Tris-buffered saline solution [pH 7.4]), supplemented with protease inhibitor cocktail (Sigma-Aldrich). The homogenate was centrifuged at 14,000g for 10 minutes at 4°C. Sample lysates were denatured at 95°C for 5 minutes in the presence of 4× LDS sample loading buffer (Invitrogen) and 5% β-mercaptoethanol (Bio-Rad). Equal amounts of protein (30 μg) were separated by 4%–20% Ready Gel Tris-HCl gels (Bio-Rad), transferred to polyvinylidene difluoride membranes, and blocked with StartingBlockT20 blocking buffer (Thermo Fisher Scientific) for 60 minutes at room temperature. Membranes were incubated with rat anti–ZO-1 monoclonal antibody (MABT 11, Sigma-Aldrich) and rabbit recombinant monoclonal anti-OCLN antibody (ab167161, Abcam) at 1:400 dilution at 4°C overnight, and they were then washed in Tris-buffered saline for 1 hour. The membranes were then probed with peroxidase-conjugated secondary antibodies at 1:8000 dilution for 1 hour at room temperature, and the bands were visualized by electrochemiluminescence (ECL, Thermo Fisher Scientific). Signals were quantified using ImageJ (NIH) and normalized to controls.

Roswell Park Memorial Institute 1640 medium (RPMI-1640), fetal bovine serum (FBS), and penicillin/streptomycin solution were purchased from Hyclone Laboratories (CA, USA). Cell counting kit-8 (CCK-8) and dimethyl sulfoxide (DMSO) were obtained from Dojindo Laboratories (Kumamoto, Japan) and Sigma-Aldrich (St. Louis, MO, USA), respectively. FITC annexin V Apoptosis Detection Kit and propidium iodide (PI) were purchased from BD Biosciences (San Jose, CA, USA). Radioimmunoprecipitation assay buffer, protease and phosphatase inhibitor cocktail, BCA protein assay kit, 4–12% bis-tris plus gels, nitrocellulose membranes, TBS Tween 20 Buffer, Starting Block T20 Blocking Buffer, enhanced chemiluminescence kit, and the following antibodies: caspase-9, cleaved caspase-9, PARP, cleaved PARP, Bcl-2, β-actin, and horseradish peroxidase (HRP)-conjugated secondary antibody were acquired from Thermo Fisher (Rockford, IL, USA). Antibodies against caspase-3, cleaved caspase-3, AMPKα, Akt, and phospho-Akt were purchased from Cell Signaling (Danvers, MA, USA).

Antibodies were purchased from commercial vendors or academic institutions that provided antibody validation information. Allophycocyanin-conjugated monoclonal antibody (mAb) against CD11b (clone M1/70, catalog 14-0112-82, Thermo Fisher Scientific) was diluted 1:25 for alveolar microinstillation. Antibodies used for immunoblotting included mouse mAb against CFTR (clone A-3, catalog sc-376683, lot I2221, Santa Cruz Biotechnology) (43 (link)); mouse mAb against dephosphorylated CFTR (clone 570, catalog AB570, lot 570TJ20200526, University of North Carolina at Chapel Hill CFTR Antibody Distribution Program) (44 (link), 45 (link)); and rabbit polyclonal antibody against actin (catalog A2066, lot 120878, MilliporeSigma). Secondary antibodies (LI-COR) included IRDye 800CW goat anti-mouse (catalog 925-32210, lot D01110-02) and IRDye 680LT goat anti-rabbit (catalog 925-68021, lot C90501-05). Antibodies were diluted in StartingBlock T20 Blocking Buffer (Thermo Fisher Scientific) and incubated with membranes as follows: CFTR mAb A-3 was diluted to 1:100 and incubated for 24–72 hours at 4°C; CFTR mAb 570 was diluted to 1:500 and incubated for 24 hours at 4°C; actin antibody was diluted to 1:2,000 and incubated for 1 hour at room temperature; and secondary antibodies were diluted to 1:10,000 and incubated for 40 minutes at room temperature.