Eclipse 2000 microscope

The expression of additional epithelial markers such as transmembrane mucin 1 (MUC1), also known as epithelial membrane antigen (EMA), and cytokeratin cocktail (AE1/AE3) were detected by immunohistochemistry methods. Cells were fixed with 4% paraformaldehyde in PBS 1X, treated with 0.1% Triton x-100 in PBS for 15 min and blocked with 2% BSA in PBS at RT for 2 h. Then, primary anti-EMA antibodies (MS-348-P, Thermo Scientific, USA) and anti-AE1/AE3 (MA5–13156, Thermo Scientific, USA) were incubated with cells at 4 °C for 16 h at 1:100 dilution. Finally, the slides were washed in PBS and incubated with an HRP conjugated goat anti-mouse IgG secondary antibody at RT for 45 min. Immunocytochemical staining was performed using an avidin-biotin complex peroxidase standard staining kit. HeLa and AGS cell lines (ATCC CRL-1739) were used as positive controls for AE1/AE3 and MUC1 markers, respectively, whereas hematopoietic U-937 cell lines (ATCC® CRL-1593.2™) were used as a negative control. Imaging was made using a Nikon Eclipse 2000 microscope (Nikon, Tokyo, Japan). All experiments were performed in triplicate.

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).