Cells were prepared onto salinized glass slides, fixed, and stained as previously described in the FISH process. Briefly, after fixation with 4% PFA, cells were washed with 0.1 M PBST pH 7.4. After, cells were incubated for 10 min with 0.1 M glycine and treated with BSA solution (Sigma) for 30 min. Cells were then incubated overnight at 4 °C with primary antibodies at 1:100 dilution in PBST and 1% BSA [SARS-CoV-2 Spike S1 antibody (#HC2001 GenScript—#A02038), p62 antibody (#BD 610832), Lamp1 antibody (#BD 555798) and CD63 antibody (#BD 556019), according to the desired double IF. The slides were washed and incubated for 2 h with secondary antibodies (Alexa 488 anti-Human IgG Thermo Fisher—#A11013 and Alexa Fluor 555 Anti-Mouse IgG #A21422), diluted 1:500 in PBST + 1% BSA. Cells were then washed and stained with DAPI (Santa Cruz Biotechnology, #SC3598). Subsequently, cells were washed with PBS, and the labels were mounted in an aqueous mounting solution for confocal imaging.
P62 antibody
Manufactured by BD
Sourced in Canada
The P62 antibody is a laboratory reagent used for the detection and analysis of the P62 protein, also known as sequestosome 1 (SQSTM1). P62 is a multifunctional protein involved in various cellular processes, such as autophagy, cell signaling, and protein trafficking. The P62 antibody can be used in techniques like Western blotting, immunohistochemistry, and immunofluorescence to identify and study the P62 protein in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Lamp1 antibody, by BD (1 mentions)
Bsa solution, by Merck Group (1 mentions)
Anti mouse igg alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Cd63 antibody, by BD (1 mentions)
Bromophenol blue, by Merck Group (1 mentions)
3 methyladenine, by Merck Group (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Cleaved caspase 9, by Santa Cruz Biotechnology (1 mentions)
P gsk3β ser9, by Santa Cruz Biotechnology (1 mentions)
P β cateninser552, by Cell Signaling Technology (1 mentions)
Histone h2b, by Cell Signaling Technology (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide, by Merck Group (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
Beclin 1, by Cell Signaling Technology (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
Fugene transfection reagent, by Promega (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Acrylamide, by Merck Group (1 mentions)
Ethidium bromide, by Merck Group (1 mentions)
Sodium dodecyl sulphate (sds), by Merck Group (1 mentions)
3 protocols using p62 antibody
1
SARS-CoV-2 Spike Protein Localization
2
Nimbolide Modulates PI3K/Akt/GSK-3β Signaling
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Acrylamide, AO, bovine serum albumin (BSA), bromophenol blue, CQ, 4,6-diamidino-2-phenylindol (DAPI), DMBA, ethidium bromide, JC-1 iodide, 3-methyladenine (3-MA), 2-mercaptoethanol, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), sodium dodecyl sulphate (SDS), N,N,N’,N’-tetramethylene diamine (TEMED) and Trizol were acquired from Sigma Chemical Company, St. Louis, MO, USA. Power SYBR® Green PCR master mix was obtained from Applied Biosystems, California, USA. Antibodies for Akt, β-actin, β-catenin, cleaved caspase-3, cleaved caspase-9, cytochrome c, GSK-3β, p-GSK-3βSer9, p-GSK-3βTyr216, PI3K, and Gapdh were purchased from Santa Cruz Biotechnology, USA. Antibodies for ATG5, Bax, Bcl-2, Beclin-1, Histone H2B, LC-3, p-AktSer473, p-β-cateninSer33,Ser37,Thr41, and p-β-cateninSer552 as well as ELISA kits were from Cell Signaling Technology, USA. Alexafluor-488 conjugated anti-rabbit antibody was obtained from Molecular Probes, Inc. (Eugene, OR, USA). Annexin V-FITC, propidium iodide (PI) kit and p62 antibody were purchased from BD Biosciences (San Diego, CA). Nimbolide was obtained from M/s Asthagiri Herbal Research Foundation, Chennai, India. FuGENE transfection reagent was procured from Promega. Oligonucleotide primers were purchased from Sigma Genosys, San Ramon, USA. All other reagents used were of analytical grade.
3
Western Blot Analysis of Protein Expression
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Upon completion of treatment, L6 cells were lysed in RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 1% Triton X and 0.5% sodium deoxycholate) containing 10% β-mercaptoethanol and phosphatase inhibitor cocktail set V (EMD Millipore), heated for 10 min in 90°C and centrifuged at 13,600 g for up to 15 min. Samples were resolved by 8-15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane, blocked in 3% bovine serum albumin (BSA) blocking buffer and then immunoblotted with primary antibodies (pPERK, peIf2α, LC3, pAKT(T308), pAKT(S473), β-actin and GAPDH purchased from Cell Signaling, pIRE1 purchased from Novus Biologicals, ATF6 purchased from Santa Cruz, p62 antibody from BD Biosciences (Mississauga, ON, Canada) and KDEL purchased from Abcam. These were subsequently detected with horseradish peroxidase (HRP)-conjugated secondary antibodies purchased from Cell Signaling. Protein bands were visualized using enhanced chemiluminescence (ECL, BioRad) reagent and quantitated by densitometry using ImageJ. All values were corrected for an appropriate loading control, β-actin or GAPDH.
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