Ds ri1 eclipse microscope

Cultured cells were fixed in 4 % (w/v) paraformaldehyde in PBS for 5 min at 37 °C. Following fixation, cells were permeabilised (0.3 % (v/v) Triton X-100 in PBS) and blocked with 3 % (w/v) bovine serum albumin before incubation with polyclonal antibodies (1 h at ambient temperature) against IRS1 (1/50; Rabbit polyclonal sc7200, SCBT), pIRS1 S616 or pIRS S636/639 (both 1/100; CST,) and a monclonal antibody against vimentin (1/200; Rabbit polyclonal ab8978, Abcam). Cells were incubated with the appropriate species of secondary antibody for 1 h at ambient temperature (1/1000; Alexa-fluor conjugated, Life Technologies) and cell nuclei were stained with Hoescht 33342 (5 μg/ml bisbenzimide in PBS). Astrocytes were examined using a Nikon DS-Ri1 Eclipse microscope (Nikon, Tokyo, Japan).

Cultured cells were fixed in 4% paraformaldehyde for 10 min at 37°C and then permeabilised in 0.3% Triton X‐100 in PBS (3 min at RT). After further washing, cells were incubated in 3% bovine serum albumin prior to incubating in occludin antibody for 2 hrs at RT (71–1,500, 1:50; Thermo Fisher Scientific). Cells were subsequently washed in PBS and then incubated with the appropriate species of secondary antibody for 2 hrs at RT (1:500 Alexa Fluor conjugated, Life Technologies). Cell nuclei were stained with Hoescht 33342 (5 μg/ml bisbenzimide in PBS). Coverslips were mounted on to glass slides using fluorescent mounting medium (DAKO) and cells were examined using a Nikon DS‐Ri1 Eclipse microscope (Nikon, Kingston, UK).

FHUS were fixed in 4% paraformaldehyde for 30 min at 25°C. Following fixation, cells were permeabilized with 0.3% (v/v) Triton X-100 in PBS and then blocked with 5% bovine serum albumin. Next, the cells were incubated with the anti-vimentin monoclonal antibody (1 : 100; cat number ab8978; Abcam, Cambridge, UK) at 37°C for 1 h and then incubated with the secondary antibody for 30 min at room temperature (1/1000; Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China). The slides were examined using a Nikon DS-Ri1 Eclipse microscope (Nikon, Tokyo, Japan).