Alexa fluor 488 conjugated goat anti rabbit

The same immunohistochemistry protocol was used for confocal immunofluorescence, but the tissue sections were incubated overnight at 4°C with the primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, United States) to P-selectin 1:200 for 1 h. After washing in phosphate buffered saline/0.2% Triton X-100 for 5 min, the tissues were then incubated with Alexa Fluor 488-conjugated goat anti-rabbit (Abcam Inc., Cambridge, MA, United States) secondary antibody (1:500) for 1 h. Fluorescent images were obtained on a Carl Zeiss laser scanning microscope (LSM 710, 20 × objective, Carl Zeiss, Jena, Germany). Tissue reactivity in all groups was assessed by computerized densitometry analysis of the digital images captured with the confocal immunofluorescence microscope. Average densitometric values were calculated in Image J software (Wayne Rasband; National Institutes of Health, Bethesda, MD, United States).

For immunocytochemical analysis of the osteoblast cells cultured on different pore size PCL scaffolds (tissue culture plate acted as the control group) for 14 and 30 days, cells were fixed with 4% paraformaldehyde in PBS (Polysciences, Warrington, PA, USA) for 30 min at room temperature then gently rinsed with PBS. The cell membranes were then permeabilized and blocked with a protein blocker solution (1% BSA, 22.52 mg Glycine in 0.1% Tween 20 in PBS), Sigma Aldrich) for 30 min. After washing, the cells were incubated in the following diluted primary antibodies at 4 °C overnight: mouse monoclonal anti-Collagen IA (1:250, SantaCruz Biotechnology, USA), rabbit polyclonal anti-Collagen III (1:100, abcam, Australia), mouse monoclonal anti-Osteocalcin (1:200, abcam, Austrailia).
The cells were rinsed in PBS (three times, 5 min per wash) and incubated in the appropriate secondary antibody i.e. Alexa Fluor 488-conjugated goat anti-rabbit (1:200, abcam, Australia) or F (ab^`)2-Goat anti-Mouse IgG FITC (1:200, ThermoFisher Scientific, USA) at room temperature in the dark for 1 h. Cell nuclei were stained using 40, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA, USA) in PBS (1:1000) for 30 min. The samples were mounted onto glass slides for visualisation using a fluorescence microscope (Nikon, Eclipse- Ti, U.S.A).

The antibodies used in this study included: mouse anti-GAPDH (Santa Cruz Biotechnology, sc-365,062), goat anti-TFE3 (Santa Cruz Biotechnology, sc-5958), rabbit anti-TFE3 (Abcam, ab93808), mouse anti-4EBP3 (Santa Cruz Biotechnology, sc-134,232), rabbit anti-pS6 (Cell Signaling Technology, 2215), rabbit anti-RPS6 (Abcam, ab40820), rabbit anti-mTOR (Cell Signaling Technology, 2972), rabbit anti-14-3-3 β/α (Cell Signaling Technology, 9636), rabbit anti-14-3-3 γ (Cell Signaling Technology, 5522), rabbit anti-14-3-3 ζ/δ (Cell Signaling Technology, 7413), rabbit anti-14-3-3 ε (Cell Signaling Technology, 9635), rabbit anti-14-3-3 τ (Cell Signaling Technology, 9638), rabbit anti-14-3-3 η (Cell Signaling Technology, 5521), rabbit anti-LAMP2 (Cell Signaling Technology, 49,067), rabbit anti-β-Tubulin (Cell Signaling Technology, 2146), HRP-conjugated goat anti-rabbit (Cell Signaling Technology, 7074), goat anti-mouse secondary antibody (Boster, BA1050), rabbit anti-goat secondary antibody (Boster, BA1060), Alexa Fluor 488-conjugated goat anti-rabbit (Abcam, ab150077), and Alexa Fluor 594-conjugated donkey anti-goat secondary antibodies (Invitrogen, A-11058).

Harvested tumors were fixed in formalin for 48 h, paraffin embedded and 5 µm thick sections were obtained. Standard hematoxylin and eosin (H&E) staining was performed. On adjacent tumor sections, immunohistochemical staining was performed. The IHC sections were deparaffinized followed by heat-mediated antigen-retrieval. Briefly, for antigen-retrieval tumor sections were hydrated and steamed for 40 min in IHC-TEK Epitope Retrieval Solution (IHC World, Ellicott City, MD). Following antigen-retrieval, tumor sections were permeabilized with methanol and were blocked using TNB Blocking buffer (PerkinElmer, Waltham, MA) for 20 min. The sections were then incubated with rabbit anti-vaccinia virus antibody diluted 1:100 in TNB Blocking buffer (Cat# ab35219; Abcam, Cambridge, MA), overnight in a humidified chamber at 4 °C. The following day, tumor sections were washed and incubated with Alexa Fluor-488-conjugated goat anti-rabbit (Cat# ab150077, Abcam, Cambridge, MA) for 1 h at room temperature. Finally, the sections were counterstained with 4′6-diamidino-2-phenylindole (DAPI) and were imaged using EVOS FL Auto Imaging System (Thermo Fisher Scientific, Waltham, MA).

Hearts were resected and fixed in 4% paraformaldehyde for at least 1 day to prepare for immunofluorescence analysis. Thereafter, the specimens were soaked in gradient sucrose solutions in PBS to dehydrate and were processed into frozen sections. Representative sections from the ischemic region were permeabilized in 0.5% Triton X-100 for 15 min and sealed with 5% BSA for 1 h. Afterwards, the sections were incubated with the following primary antibodies at 4°C for 12 h: anti-COX2 (1:100, Cat no. ab15191; Abcam), anti-NLRP3 (1:100, Cat no. NBP2-12446; Novus, Littleton, CO, United States), anti-Vimentin (1:100, Cat no. 5741; CST) and anti-Smooth Muscle Actin (1:100, Cat no. sc-53142; Santa Cruz Biotechnology, Santa Cruz, CA, United States). Primary antibodies were detected by Alexa Fluor 488-conjugated goat anti-rabbit (1:500, Cat no. ab150077; Abcam), Alexa Fluor 568-conjugated goat anti-rabbit (1:500, Cat no. ab175471; Abcam), or Alexa Fluor 488-conjugated goat anti-mouse (1:500, Cat no. ab150113; Abcam) secondary antibodies, respectively, for 2 h at room temperature. Nuclei were identified with its marker of 4′,6-diamidino-2-phenylindole (DAPI) (Cat no. 62248; Thermo Fisher Scientific). Finally, the sections were observed with a fluorescence microscope.