Secondary goat anti mouse igg polyclonal antibodies conjugated with alkaline phosphatase

The Western blot method was used to evaluate the expression of JAK3, STAT2, STAT4 and STAT6. Total protein from frozen skin samples from PV, BP, CUS and LP patients and healthy controls were extracted in RIPA protein extraction buffer, supplemented with protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). The lysate was centrifuged and the pellet was discarded. Protein concentrations were determined by the BCA Protein Assay Kit (Pierce Thermo Scientific, Waltham, MA, USA) according to manufacturer’s instructions.
The membrane was blocked with non-fat milk in TBST and then incubated with the mouse primary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA). Afterwards, the membrane was incubated with secondary goat anti-mouse IgG polyclonal antibodies conjugated with alkaline phosphatase (Santa Cruz Biotechnology, Dallas, TX, USA). The bands were developed using BCIP/NBT Alkaline Phosphatase Substrate (Merck Millipore, Darmstadt, Germany), analyzed using the ImageJ 1.34s software (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA), which allowed for image analysis of densitometry expressed as % optical density (OD) over the background. Obtained results were expressed as the mean ± SD.

Western blot method was used to evaluate the expression of JAK1, JAK2, STAT1, STAT3, and STAT5 in skin lesions of DH and BP groups (and not perilesional skin) and compared with healthy control group. STAT5 antibody recognized both STAT5a and STAT5b.
Total proteins from frozen skin samples from BP and DH patients and healthy controls were extracted in RIPA protein extraction buffer, supplemented with protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). The lysate was centrifuged, and the pellet was discarded. Protein concentrations were determined by the BCA Protein Assay Kit (Pierce Thermo Scientific, USA) according to manufacturer's instructions.
The membrane was blocked with nonfat milk in TBST and then incubated with the mouse primary antibodies (Santa Cruz Biotechnology, Dallas, USA). Afterwards, the membrane was incubated with secondary goat anti-mouse IgG polyclonal antibodies conjugated with alkaline phosphatase (Santa Cruz Biotechnology, Dallas, USA). The bands were developed using BCIP/NBT Alkaline Phosphatase Substrate (Merck Millipore, Darmstadt, Germany) and analyzed using the ImageJ 1.34s software (Wayne Rasband, National Institutes of Health, Bethesda, MD), which allowed for image analysis of densitometry expressed as % optical density (OD) over the background. Obtained results were expressed as the mean ± SD.