Cells were harvested in LIPA buffer (Sigma) as previously reported13 (link). After centrifugation at 15,000 × g for 5 min, the supernatant was boiled for 5 min in SDS-PAGE sample buffer (Wako) and loaded onto 5–20% gradient or 15% gels for 1~2 h. Proteins were transferred to nitrocellulose membranes (Bio-Rad) by using the Trans-Blot Electrophoretic Transfer cell (Bio-Rad). The membrane was washed in 20 mM Tris-HCl buffer with 1% Tween 20 (TBS-T) (Sigma-Aldrich), incubated for 1 h in blocking buffer (5% milk powder (Merck) in TBS-T), and then further incubated overnight with primary antibodies; Anti-LC3 (Cell signaling) and anti-GAPDH (Santa Cruz) antibodies were purchased and diluted in blocking buffer by 1:3000. The horseradish peroxidase (HRP)-labeled secondary antibody (Dako) was diluted by 1:5000 in TBS-T for secondary antibody staining for 1 h at RT. The membrane was then washed in TBS-T and the signals were developed using the Lumi-Light reagent (Roche) for 5 min. The signal was detected on the LAS-3000 imaging system (Fuji).
Horseradish peroxidase hrp labeled secondary antibody
Manufactured by Agilent Technologies
Sourced in Denmark
Horseradish peroxidase (HRP)-labeled secondary antibody is a laboratory reagent used for immunodetection. It consists of a secondary antibody conjugated with the enzyme horseradish peroxidase. This conjugate can be used to detect and visualize target proteins in various immunoassay techniques, such as Western blotting, ELISA, and immunohistochemistry.
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Lab products found in correlation
Las 3000 imaging system, by Fujifilm (1 mentions)
Lumilight reagent, by Roche (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Tbs t, by Agilent Technologies (1 mentions)
Milk powder, by Merck Group (1 mentions)
Tbs t, by Merck Group (1 mentions)
Tbs t, by Roche (1 mentions)
Tween 20 tbst, by Merck Group (1 mentions)
Trans blot electrophoretic transfer cell, by Bio-Rad (1 mentions)
Sds page sample buffer, by Fujifilm (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
Cd3 clone sp7, by Abcam (1 mentions)
Cd8 clone 144b, by Abcam (1 mentions)
2 protocols using horseradish peroxidase hrp labeled secondary antibody
1
Western Blot Analysis of LC3 Protein
2
Immunohistochemical Analysis of T-cell Subsets
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Standard immunohistochemical analysis was performed against primary antibody directed CD3 (clone SP7, diluted at 1: 100, Abcam, Cambridge, UK), CD4 (clone B468A1, diluted at 1: 200, Santa Cruz, Texas, USA) and CD8 (clone 144B, diluted at 1: 100, Abcam, Cambridge, UK). Antigen retrieval was achieved using EDTA buffer (pH 9.0) in a pressure cooker for 20 minutes. After neutralization of endogenous peroxidase, tissue microarray slides were pre-incubated with blocking serum and then were incubated with primary antibody for 45 minutes at room temperature. After three five-minute washes with PBS, the slides were treated with the horseradish peroxidase (HRP)-labeled secondary antibody (Dako, Glostrup, Denmark) for 20 minutes at room temperature, then washed in PBS. Finally, the reaction products were visualized with 3,3′-diaminobenzidine (DAB, Dako, Glostrup, Denmark) and the slides were counterstained with hematoxylin. After being dehydrated, slides were mounted in resin.
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