Coralite594 conjugated goat anti rabbit igg h l

1 × 10⁵ cells were seeded on cell climbing slices coated with poly-D-lysine in 24 well plates. Prepared cells were fixed in 4% PFA for 20 min, permeabilized with 0.5% Triton X-100 for 5 min, and blocked in 10% goat serum for 1 h at room temperature. The following primary antibodies were incubated overnight at 4 °C: anti-LDHA (1:250, Proteintech), anti-β-catenin (1:200, Proteintech), or anti-HIF-1α (1:200, Abcam; 1:100 Proteintech). Next day, cells were incubated with secondary antibodies for 1.5 h at room temperature: CoraLite594-conjugated goat anti-rabbit IgG(H + L) (1:250, Proteintech), CoraLite488-conjugated goat anti-mouse IgG(H + L) (1:250, Proteintech). Cell climbing slices were mounted on slides with mounting medium containing DAPI and photographed by a TCS SP8 confocal laser scanning microscopy (Leica, Italy) or an Axio Scope A1 microscope (Zeiss, Germany) at 400× magnification.

The fixation of HPMECs was performed using 4% paraformaldehyde followed by permeabilization using 1% Triton X-100 (Biosharp, China, BS084), followed by blocking for 10 min with quick blocking buffer (Beyotime, China, P0260) at room temperature. The paraffinized lung tissue sections were dewaxed, and antigen repair was performed, followed by blocking. Then, cells and lung tissues were incubated with Nrf2 (Proteintech, China, #16396-1-AP, 1:300) and p-p65 (CST, USA, #3033, 1:1600) primary antibodies for 10–16 h at 4 ^∘C. After washing the excess primary antibodies the next day three times with pre-cooled PBS for 10 min, the tissue samples were incubated with the respective fluorescent CoraLite594-conjugated Goat Anti-Rabbit IgG (H+L) (Proteintech, China, #SA00013-4, 1:300) secondary antibodies. The mixture was then stained with an antifade mounting medium for fluorescence (with DAPI) (Biosharp, China, BL739A). The cells and tissues were then observed and photographed under laser confocal microscopy.

To evaluate the inflammation and epidermal regeneration in the wound area, tissues containing the wound site and their surrounding healthy skin were collected. The tissue samples were fixed in 4% (v/v) paraformaldehyde for 1 h right after sacrifice before embedded in paraffin. The samples were cross sectioned to slices (4 μm thickness) and then stained by Hematoxylin-Eosin (H&E). All slides were scanned and analyzed by a Digital Slide Scanner (KFBIO, Ningbo). The regenerated skins from the wound site were also excised for IF staining with Anti-VEGFA antibody (proteintech) and TNFA Ab (proteintech), respectively. CoraLite488-conjugated Affinipure Goat Anti-Mouse IgG(H + L) (proteintech) and CoraLite594-conjugated Goat Anti-Rabbit IgG(H + L) (proteintech) were used as the secondary antibody to reveal VEGFA and TNFA expression. The nuclei were stained with 4′,6-diamidino-2-phenylindole. Slides were observed under an upright fluorescence microscope (BX53, Olympus).

Briefly, cells were fixed with 4% paraformaldehyde for 30 min, followed by permeabilization with 0.3% Triton X-100 for 15 min at room temperature. Then, the cells were incubated with rabbit anti-ORF2 polyclonal antibody (1:2,000) overnight at 4°C, followed by incubation with CoraLite594-conjugated goat anti-rabbit IgG(H+L) (Proteintech, SA00013-4, 1:500) at 37°C for 1 h. Finally, cellular nuclei were stained with Hoechst 33342 (Thermo Fisher, H1399, 1:500) for 10 min at room temperature. After washing three times with PBST buffer, cells were visualized using an florescence microscope (Olympus).

Cells were inoculated in a 12-well plate and fixed with 4% paraformaldehyde (Servicebio, Cat No. G1101, Wuhan, China) for 20 min. After washing, cells were sealed with 5% bovine serum albumin (Sigma-Aldrich, Cat No. A1933, USA) for 1 h, followed by incubation overnight with primary antibody at 4 °C (see Table S3 for specific antibodies). Next, cells were washed three times and incubated with CoraLite 594-Conjugated Goat Anti-Rabbit IgG (H+L) (Protein Tech, Cat No. SA00013-4, 1:100 dilution, Wuhan, China) at room temperature for 1 h. The nuclei were stained with DAPI (Beyotime, Cat No. C1006, Shanghai, China) and incubated at room temperature for 10 min. Anti-fluorescence quenching sealing solution (Beyotime, Cat No. P0126, Shanghai, China) was used to seal the film for 5 min. Images were taken under a fluorescence microscope (Olympus IX73, Tokyo, Japan).

After behavioral testing, the rats were anesthetized with an intraperitoneal injection of pentobarbital sodium. Following cardiac perfusion with 0.9% sodium chloride and 4% paraformaldehyde, the brains were extracted, fixed in 4% paraformaldehyde for 24 h and dehydrated using an alcohol gradient. Coronal paraffin sections with a thickness of 3 μm were obtained from paraffin blocks using a microtome. Subsequent to high-temperature repair and blocking, the sections underwent an overnight incubation at 4°C with the primary antibody, including anti-phospho-TAK1(Thr184/187) (rabbit. # PA5-99340; Thermo Fisher Scientific), anti-Neun (rabbit. # 26978-1-AP; Proteintech), anti-IBA1(mouse. # ab283319; Abcam), anti-glial fibrillary acidic protein (GFAP; mouse. # 60190-1-ig; Proteintech), and anti-GSDMD (rabbit. # 20770-1-AP; Proteintech). After three washes in phosphate buffer solution (PBS), the sections were exposed to appropriate conjugated secondary antibodies: CoraLite488-conjugated Affinipure Goat Anti-Mouse IgG([H+L] SA00013-1; Proteintech) and CoraLite594-conjugated Goat Anti-Rabbit IgG([H+L] #SA00013-4; Proteintech), followed by counterstaining with DAPI for 10 min in the dark. Images were captured using a fluorescence microscope.

The cells were fixed in 4% paraformaldehyde for 15 min and then permeabilized in 0.3% Triton X-100 for 15 min. Subsequently, the cells were blocked with blocking solution (3% bovine serum albumin (BSA), 0.3% TritonX-100, 10% FBS complemented with PBS) for 2 h. Then, Anti-MyHC (1:1000; Millipore, Billerica, MA, USA) or anti-MyoG (1:500; Abclonal, Wuhan, China, Cat#A17427) were added and the solution was incubated overnight at 4 °C. After that, the cells were stained with CoraLite594-conjugated Goat Anti-Rabbit IgG (H + L) (Proteintech, Wuhan, China, Cat#SA00013-4) for 1 h. The cell nuclei were stained using DAPI (Servicebio, Wuhan, China, Cat#G1012-10ML) solution in darkness for 10 min. Images from three randomly selected fields were obtained with a Leica SP8 confocal microscope and processed with Image J software (version 1.53e).