Ba1032

Prepared slices were washed in PBS for 10 min and then boiled in a 0.01 mmol citrate buffer (pH = 6) for 10 min for antigen retrieval. After incubation with hydrogen peroxide for 10 min, 5% bovine serum albumin (BSA) was applied as the blocking solution for 20 min at room temperature. Without being washed, the sections were incubated with anti-p-Akt (1:200; #4060, Cell Signaling Technology, MA, USA) or anti-p-Bad (1:200; sc-12969-R, Santa Cruz, CA, USA) antibodies overnight at 4 °C. After being rinsed with PBS, the sections were incubated with FITC (1:50; BA1105, Boster, Wuhan, China)- or Cy3 (1:50; BA1032, Boster, Wuhan, China)-labeled goat anti-rabbit secondary antibodies for 2 h at 37 °C in the dark. The sections were rinsed and stained with DAPI (100 ng/ml; Boster, Wuhan, China) for 8 min at room temperature and then mounted with Vectashield mounting medium (Boster, Wuhan, China). All slices were observed and photographed under a fluorescence microscope (DM5500B, Leica, Solms, Germany).

Tissue sections of 5-μm thickness were cut from frozen glioblastoma specimens and processed for immunofluorescence. Sections were immunostained with rabbit anti-ATF5 (Abcam, ab60126) and mouse anti-IE (Virostat-Inc, 0841) antibodies and then stained with anti-rabbit IgG conjugated with CY3 (boster, BA1032), and anti-mouse IgG conjugated with FITC (boster, BA1101). Following repeated washes in PBS, nuclei were counterstained with Hoechst dye. The images were obtained using Olympus FV1200 fluorescencemicroscope.

Trophoblast cells were fixed in 4% paraformaldehyde solution for 15 min and blocked in 1% BSA in PBS for 30 min, followed by incubation overnight at 4°C with a primary antibody against Fyn (1 : 1000, Ab125016, Abcam, Cambridge, MA, USA). Afterwards, Cy3-conjugated secondary antibodies (1 : 1000, BA1032, Boster, Wuhan, China) were added for 1 hr at 37°C. The cell nuclei were stained with 4,6-diamino-2-phenyl indole (DAPI) (C1002, Beyotime Biotechnology, Shanghai, China) for 5 min, and the images were captured using a fluorescence microscope (BX53, Olympus, Tokyo, Japan).

Animal models of AHR induced by RSV were established according to the previous method [22 (link)]. Briefly, 6-8 weeks BALB/c female mice weighing 16–20 g (purchased from Hunan Tianqin Biological Technology Co., Ltd., Changsha, China) were kept in a pathogen-free environment. The mice were randomly divided into the control group (n = 12) and the RSV group (n = 12). After anaesthetizing the mice with isoflurane, 5 × 10⁶ pfu RSV in 100 μL, was intranasally inoculated. For mock infections, mice were given an equivalent volume of sterile PBS. The airway resistance of mice was tested on day 7 and 28 post-infection (6 mice from each group). The mice were then sacrificed, and their lungs were taken for follow-up studies. RSV infection was verified by indirect immunofluorescence (IFA) with RSV major surface glycoprotein G monoclonal antibody (Bioss, bs-1264R, Beijing, China) as the primary antibody and CY3-conjugated antibody (Boster, BA1032, Wuhan, China) as the secondary antibody.

Lung tissue sections were fixed with 95% ethanol and 0.1% Triton-X100. Samples were then blocked with normal goat serum for 20 min and incubated overnight with RSV major surface glycoprotein G monoclonal antibody (Bioss, bs-1264R, Beijing, China) rabbit primary antibody at 4 °C. Then, the samples were incubated with a CY3-conjugated goat anti-rabbit secondary antibody (Boster, BA1032, Wuhan, China) at room temperature for 1 h. After being counterstained with DAPI for 10 min, samples were observed under a light microscope at high magnification (200×) (Leica, Wetzlar, German).

U87 and shATF5-U87 cells were maintained in serum free medium and were trypsinized and replated onto glass coverslips in 12-well tissue culture plates. After infection, cells on the glass coverslips were first fixed with buffered 4% paraformaldehyde, pH 7.4, for 30 min at room temperature and then permeabilized with 0.1 M PBS containing 0.2% Triton X-100 for 30 min and thereafter preincubated with 5% normal goat serum for 30 min prior to incubation with the primary antibodies at 4°C for overnight. The primary antibodies employed were against the antigens ATF5 (diluted 1:1000; Abcam) and immediate-early (IE) protein (diluted 1:100; Virostat-Inc). After thorough rinsing with 0.1 M PBS, slides were incubated with the secondary antibody conjugated CY3 (diluted 1:500; boster, BA1032) or FITC (diluted 1:200; boster, BA1101). Following repeated washes in PBS, nuclei were counterstained with Hoechst dye. To ensure antibody specificity, negative control slides were processed in the same manner, but omitting the primary antibodies. The images were obtained using Olympus FV1200 fluorescencemicroscope.