Goat anti rabbit igg alexa fluor 647

Detection of cellular proteins was performed using immunofluorescence as described previously [33 (link)]. Cells were cultured on 180-μm-thick polymer coverslip 8-well plates (μ-Slide ibiTreat plates #80826) and treated as appropriate. After 24 h, cells were fixed using 4% paraformaldehyde. Cells were permeabilized using 0.1% Triton in PBS, blocked using Odyssey blocking buffer, and incubated with primary antibody overnight at 4 °C. Primary antibody binding was detected using AlexaFluor 488 goat anti-mouse IgG (Abcam 1:500); AlexaFluor 647 goat anti-rabbit IgG (1:500); or AlexaFluor 568 goat anti-goat IgG (Abcam, 1:500). Cells were stained with DAPI and mounted using Prolong^TM Gold antifade reagent (Invitrogen, #P36934). Slides were imaged using a Wave FX spinning disc confocal microscope (Zeiss) with Volocity 6.3 acquisition and analysis software (Perkin Elmer), and basic contrast enhancement performed using unbiased automatic black-point calculation. Composite z-stack images included 15-20 XY planes over a total vertical distance of 4–6 μm using the Improvision Focus Drive. All cells were imaged using a × 40 oil immersion objective lens and quantification was performed on a single XY plane per field of view.

Embryos were fixed in 4% PFA overnight at 4°C and sectioned as described above. Sections were rehydrated in PBS for 30 min and blocked with 5% goat serum/PBS for 1 hr at room temperature. Primary antibody incubation was done overnight at 4°C. Secondary antibody incubation was done for 2 hr at room temperature. Antibodies used were: chicken anti-GFP (1:500; abcam ab13970), mouse anti-GFAP (1:1000; ZIRC zrf-1), rabbit anti-PH3 (1:2000; Millipore 06–570), rabbit anti-BLBP (1:1000; Millipore ABN14), rabbit anti-Sox2 (1:500; abcam ab97959), Alexa Fluor 647 goat anti-rabbit IgG(H+L) (1:1000), Alexa Fluor 488 goat anti-chicken IgG(H+L) (1:1000), Alexa Fluor 647 goat anti-mouse IgG(H+L) (1:1000).

The histological sections with hematoxylin-eosin (HE), Masson, and immunohistochemistry staining were employed to analyze the regeneration of skeletal muscle tissue according to reported literature [45 (link), 46 (link)]. The tibialis anterior muscles with the defect site were harvested and fixed in 4% formalin then embedded in paraffin. The cross-section of the central area of each sample was sliced using a microtome (Leica, Hamburg, Germany) and stained following the procedure of hematoxylin-eosin staining kit (Invitrogen) and Masson staining kit (Abcam). The regenerated tissues and fibrosis were observed using an optical microscope (Olympus). Immunohistochemistry was performed using the primary anti-mouse or anti-rabbit antibodies against GFP, vWF, and CD68 (1:100, all purchased from Abcam), followed by goat anti-mouse IgG Alexa Fluor 596 or goat anti-rabbit IgG Alexa Fluor 647 (1:400, Abcam). Finally, the sections were stained with Hoechst (1:1000, Abcam) prior to be imaged by fluorescent microscope (Olympus). The quantitative results were analyzed using ImageJ software with three randomly selected fields of each section and three sections per specimen.

Immunofluorescence was performed as previous described by Gyamfi et al. Briefly, cells are seeded on cover slide placed in co-culture insert and co-cultured with adipocytes for 48 h. Cells was rinsed in PBS and fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 and stained with appropriate primary antibodies. For double staining experiments, antibodies were diluted together and incubated with cells overnight at 4 °C. Goat Anti-Rabbit IgG (Alexa Fluor 647) and Goat Anti-mouse IgG (Alexa Fluor 488) antibodies (Abcam) were used as secondary antibodies. Counter staining of cell nuclei was performed using DAPI (Invitrogen, Carlsbad, CA, USA). Stained cells were visualised using the ZEISS LSM 710 microscope (ZEISS, Germany). Antibodies used included mouse anti-E-cadherin 1:500 (Cell Signalling, Danvers, MA, USA), rabbit anti-Vimentin 1:500 (Proteintech).

The cells were seeded into 4-well Millicell EZ SLIDE^® (Merck KGaA) plates and cultured until they were 70% confluent. After three washes with PBS, the cells were fixed with 4% formaldehyde (Wuhan Guge Biotech Ltd., Hubei, China) for 30 minutes in the dark and then washed three times with PBS and permeabilized with 0.1% Triton X-100 (Sangon Biotech Corp.) for 10 minutes. The cells were washed three times with PBS and blocked with 5% BSA in PBS for 1 hour. All steps were performed at room temperature. Then, the fixed cells were incubated with the specific primary antibodies listed in Supplementary Table S2 overnight in 4 °C, and the slides were incubated with goat anti-rabbit IgG (Alexa Fluor^® 647) (1:400, Abcam, Cambridge, UK) for 2 hours at room temperature in the dark. The cell nuclei were counterstained with DAPI (Beyotime Biotech Co., Ltd.) for 10 minutes. The slides were washed three times between each step of antibody incubation. The negative controls were processed in the same manner, except that the primary antibodies were omitted. The cells on the slides were observed and imaged using a confocal laser scanning microscope (Zeiss LSM710, Carl Zeiss AG, Germany).

BEAS-2B cells and MLE-12 cells were cultured on sterile coverslips in 12-well plates overnight. The cells were fix with 4% paraformaldehyde for 15 min and permeabilized in 0.5% Triton X-100 for 30 min at room temperature. Then, the cells were blocked with normal goat serum (ORIGENE, SP-9000, Beijing, China) for 30 min at room temperature and incubated with anti-NF-κB antibody (SANTA CRUZ, Dallas, TX, USA, #SC-8008) overnight at 4 °C. The cells were washed 3 times with PBS and then incubated with goat anti-rabbit IgG Alexa Fluor^® 647 (Abcam, ab150115, Cambridge, UK) in the dark for 1 h at 37 °C. A fluorescent sealing liquid solution, containing DAPI was added to mark nuclei. The images were observed and collected using a fluorescence microscope (Invitrogen EVOS™ FL Auto 2 Imaging System, AMAFD2000, Thermofisher, Waltham, MA, USA).