Ab194356

IHC was performed on 6 μm sections of formalin-fixed, paraffin-embedded tissues. The sections were deparaffinized and dehydrated, and antigen retrieval followed. Then, the sections incubated with primary antibody toward CD31 (AF806, R&D Systems), CAVIN2 (NBP1-44090, Novus), HSPG2 (AF2364, R&D Systems), MYO1B (ab194356, Abcam), ABCG2 (ab24115, Abcam), SLC2A1 (HPA058494, Sigma-Aldrich), ABCB1 (HPA002199, Sigma-Aldrich), TJP1 (HPA001637, Sigma-Aldrich), CLDN5 (341600, Invitrogen), and PLVAP (NBP1-83911, Novus). All quantification was done by 2 individual scientists in a blinded fashion.

After H/R irritation and/or OE-Myo1b (or si-Myo1b) transfection, total proteins of H9c2 cells were detached using RIPA Lysis Buffer (Beyotime Biotechnology). BCA assay (Beyotime Biotechnology) was performed to measure protein concentration. Then, proteins in equal concentration were electrophoresed on polyacrylamide gels and transferred onto polyvinylidene fluoride (PVDF) membrane, which were incubated with anti-Myo1b antibody (ab194356, 1 : 1000, 60 min), anti-Bcl-2 antibody (ab196495, 1 : 1000, 40 min), anti-Bax antibody (ab32503, 1 : 1000, 40 min), anti-LC3B antibody (ab63817, 1 : 500, 40 min), anti-p62 antibody (ab91526, 1 : 1000, 60 min), anti-GAPDH antibody (ab8245, 1 : 10000, 40 min, Abcam Biotechnology, CA, USA), or anti-cleaved-caspase 3 antibody (#9661, 1 : 1000, 60 min, Cell Signaling Technology, MA, USA). Then, PVDF membranes were incubated with HRP goat anti-mouse IgG (BA1051, 1 : 10000) or HRP goat anti-rabbit IgG (BA1054, 1 : 10000, Boster Biological Technology, Wuhan, China) for 60 min. Results were visualized via enhanced chemiluminescence technique. Intensities of proteins were analyzed via the Image-Pro Plus 6.0 software.

An NB FFPE (formalin-fixed paraffin-embedded) tissue microarray we recently described (64 (link)) was used in this study. MYO1B and NCAM expression in the FFPE tissues was assessed using a Ventana DISCOVERY Ultra autostainer (Ventana Medical Systems, Tucson, AZ). For MYO1B IHC, baked and deparaffinized FFPE tissue sections were first incubated in tris-based buffer (CC1, Ventana) at 95°C for 1 hour for antigen retrieval, followed by incubation at room temperature for 1 hour with MYO1B antibody (1:500, Abcam, ab194356) or NCAM1 antibody (1:200, Abcam, ab133345) prepared in DISCOVERY Ab diluent (Ventana). Bound primary antibodies were amplified with AffiniPure Goat Anti-Rabbit IgG (H+L) (1:500, Jackson ImmunoResearch, rabbit polyclonal, catalog no. 111-005-003) and visualized with the UltraMap DAB anti-Rb Detection Kit (Ventana). For IHC scoring, staining intensity was assigned via a four-point scale system (0 = no staining, 1 = low, but detectable degree of staining, 2 = clearly positive cases, and 3 = strong expression) and percentage of positive cells (0 to 100%) was also determined. IHC H-score was then calculated per sample as staining intensity multiplied by percentage of positive cells. For analysis of tissue microarrays (TMAs), the average IHC score was calculated among duplicate tissue cores from the same patient.