Goat anti rabbit alexa 594

Immunofluorescent staining was performed on LAM patient lung tissue [18 (link)] or 621-102 TSC2-null cells. Lung tissue slides (10 μm) were deparaffinized using standard protocols for immunohistochemistry. 621-102 Tsc2-null cells were grown on glass coverslips and treated as for the 6-well immunoblot procedure, then fixed with 4% PFA, washed with PBS, and blocked with 1% BSA, 30 min before immunostaining. For antigen retrieval (peIF4E and IgG), slides were boiled 20 min in citrate buffer with 0.05% Tween 20 (pH 6.0) and then blocked for 20 min with 1% BSA. All primary antibodies were used at a 1:250 dilution. The cells were incubated in primary antibodies, namely pS6 (Ser235/236) or α-SMA (Cell Signaling, Danvers, MA, USA) or peIF4E (Ser209) Abcam (76256), for 1 h at RT. Lung tissue slides were incubated in primary antibody, overnight at 4 °C. Secondary antibodies were goat anti-mouse Alexa-594 (Abcam, #150116), goat anti-rabbit Alexa-594 (Abcam, #150080), and goat anti-rabbit Alexa-488 (Abcam, #150077). All secondary antibodies were used at 1:250 dilution. Cell or tissue slides were incubated with appropriate secondary antibody for 1 h at RT, then stained with DAPI (10 mM) (Sigma-Aldrich Gaithersburg, MA, USA) for 10 min, washed, and embedded in 80% glycerol. Samples were analyzed and pictures were taken with a Nikon Eclipse 2000 microscope (Nikon, Melville, NY, USA).