The lung paraffin sections were dewaxed, hydrated, antigen-repaired, and circled. The paraffin-embedded sections or primary cell slides were sealed with 10% goat serum at room temperature (22–24 °C) for 1 h, then away from light at 4 °C overnight with antibodies:anti-sox9 (1:100, Santa Cruz), anti-β-catenin (1:200, Proteintech), anti-SPC (1:100, Proteintech), and anti-AQP5 (1:100, Proteintech). Next, the goat anti-rabbit or goat anti-mouse secondary antibody (1:200, Proteintech) was used to incubate for 2 h. After DAPI was used to stain the cell nucleus, a fluorescence microscope (E800, Nikon, Japan) was used to observe protein expression.
Anti spc
Manufactured by Proteintech
Sourced in China
The Anti-SPC product is a laboratory reagent designed for use in research applications. It serves as a detection tool for specific target proteins. The core function of this product is to provide a reliable and accurate means of identifying the presence of the target proteins within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Anti aqp5, by Proteintech (2 mentions)
Anti β catenin, by Proteintech (2 mentions)
Anti sox9, by Santa Cruz Biotechnology (2 mentions)
Anti β actin, by Proteintech (2 mentions)
Goat anti rabbit or goat anti mouse secondary antibody, by Proteintech (1 mentions)
Enhanced chemiluminescence detection reagent, by Merck Group (1 mentions)
Anti claudin 4, by Proteintech (1 mentions)
Protease and phosphatase inhibitors, by Biosharp (1 mentions)
Anti pten, by Abcam (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Proteintech (1 mentions)
Anti bcl 2, by Proteintech (1 mentions)
Anti keratin 8, by Proteintech (1 mentions)
Anti p akt, by Proteintech (1 mentions)
β actin, by Proteintech (1 mentions)
Anti akt, by Proteintech (1 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (1 mentions)
Anti bax, by Abcam (1 mentions)
Anti mouse hrp, by Proteintech (1 mentions)
Enhanced chemiluminescence western blotting detection system, by Merck Group (1 mentions)
Ripa lysis buffer, by Transgene (1 mentions)
4 protocols using anti spc
1
Immunofluorescence Analysis of Lung Cells
2
Quantification of Lung Protein Markers
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Total proteins were extracted from lung tissues using a radioimmunoprecipitation assay buffer (Beyotime, China) supplemented with protease and phosphatase inhibitors (Biosharp, China). Their levels were quantified using a bicinchoninic acid kit. Proteins were separated with sodium dodecyl sulfate–polyacrylamide gel and transferred to polyvinylidene fluoride membranes. They were blocked in 5% skim milk at 37 °C for 1 hour and incubated overnight at 4°C with primary antibodies: anti-PTEN (1:2000; Abcam, USA), anti-AKT (1:4000; Proteintech, China), anti-p-AKT (1:4000; Proteintech, China), anti-Bcl-2 (1:1000; Proteintech, China), anti-Bax (1:2000; Abcam, USA), anti-Cleaved Caspase 3 (1:2000; CST, USA), anti-SPC (1:1000; Proteintech, China), anti-AQP5 (1:1000; Proteintech, China), anti-Claudin 4 (1:4000; Proteintech, China) and anti-Keratin 8 (1:4000; Proteintech, China). β-actin (1:10000; Proteintech, China) was used as a reference protein. The membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1:10000; Proteintech, China) at 37 °C for 1 hour. Protein bands were detected with enhanced chemiluminescence detection reagents (Millipore, USA) and analyzed using ImageJ software (version 1.51; National Institutes of Health, USA).
3
Protein Expression Analysis of Lung Cells
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Cells or tissue were lysed in RIPA Lysis Buffer (TransGen Biotech, Beijing, China) contained with 1% Phenylmethanesulfonyl fluoride (TransGen Biotech). Proteins (30 μg/lane) were separated on 10% SDS-polyacrylamide gradient gels and transferred onto PVDF membranes. The protein-free rapid sealing fluid (Epizyme, Shanghai, China) was used to block non-specific binding, and membranes were probed with primary antibodies in 4 °C for 12 h(anti-SOX9 (1:500, Santa Cruz, Santa Clara, CA, USA), anti-β-catenin (1:10 000, Proteintech, Shanghai, China), anti-SPC (1:500, Proteintech), anti-AQP5 (1:500, Santa, USA), and anti-β-actin (1:20 000, Proteintech)), followed by incubation with anti-rabbit-HRP(1:5000; Proteintech) or anti-mouse-HRP (1:5000; Proteintech) in 37 °C for 2 h. β-actin was selected as the internal reference. The protein bands were visualized with the enhanced chemiluminescence western blotting detection system (Millipore, Billerica, MA, USA).
4
Immunoblotting Analysis of Lung Proteins
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The total protein were extracted using Radioimmunoprecipitation Assay (RIPA) lysis buffer supplemented with protease and phosphatase inhibitor cocktails (Applygen, C1055, Beijing, China). Protein concentration was measured with a bicinchoninic acid (BCA) protein assay kit (Applygen, P1511). Protein was separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Immunoblotting was performed using specific antibodies, including anti-MED19 (Invitrogen, PA5-78656, Carlsbad, CA, USA), anti-α-SMA (Proteintech), anti-SP-A (Proteintech, 11850-1-AP), anti-SP-B (Bioss, bs-22341R, Beijing, China), anti-SP-C (Proteintech, 10774-1-AP), anti-SP-D (Proteintech, 11839-1-AP), anti-P53 (CST, 2524S, Danvers, MA, USA), anti-B-cell lymphoma-2 (Bcl-2) (PTM Bio., PTM-5777, Hangzhou, China), anti-β-actin (Proteintech, 20536-1-AP), and anti-α-tubulin (PTM Bio., PTM-5442). The protein bands were visualized with a Chemiluminescent HRP Substrate (Millipore) using an automatic chemiluminescence imaging system (Tanon, Shanghai, China). The band intensity was quantified using ImageJ software, version 1.52 (National Institutes of Health) and normalized to α-tubulin or β-actin.
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