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Subconfluent HaCaT and HFs grown on coverslips were treated with rNPM. Cells were fixed 30 min with 4% paraformaldehyde in PBS and permeabilized for 5 min with 0.1% Triton X-100. The following primary antibodies diluted in 1% BSA were incubated for 1 h at 37°C in a humid chamber: α-p65rel NF-kB (1:200; F6 Santa Cruz). Mouse IgG isotype control (Santa Cruz) was used at the same concentration of primary antibody. After PBS washes, secondary antibodies goat α-rabbit IgG-Texas Red (1:200 in PBS; Jackson Immunoresearch Laboratories, West Grove, PA, USA) were added and incubated for 1 h at 37°C in a humid chamber. Nuclei were stained with DAPI (1:10,000 in PBS; Sigma). Images were acquired with the ApoTome System (Zeiss) connected with an Axiovert200 inverted microscope (Zeiss); image analysis was performed with ZEN software (Zeiss).

Subconfluent hCmPCs grown on coverslips were treated with either Dox or rNPM. Cells were fixed 30 min with 4% paraformaldehyde in PBS and permeabilized 5 min with 0.1% Triton X-100. The following primary antibodies diluted in 1% BSA were incubated 1h at 25°C: NPM1 (1:400 Abcam ab86712), γH2AX (1:400; 05-636Millipore), α-TLR4 (1:100 in PBS; Santa Cruz), and α-p65rel NFkB (1:200; F6 Santa Cruz). After PBS washes, secondary antibodies were added 1h at R.T.: goat α-mouse IgG-FITC and goat α-rabbit IgG-Texas Red (1:200 in PBS; Jackson Immunoresearch Laboratories, West Grove, PA, USA). Nuclei were stained with DAPI (1:10,000 in PBS; Sigma). Images were acquired with the ApoTome System (Zeiss) connected with an Axiovert200 inverted microscope (Zeiss); image analysis was performed with ZEN software (Zeiss).