Alexa fluor 488 goat anti rabbit igg

After deparaffinizing the sections of each group, we performed antigen retrieval with the agent ImmunoSaver Antigen Retriever (Electron Microscopic Sciences, Hatfield, PA) and blocking with 1% bovine serum albumin. Immunofluorescence staining was performed using ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs); -5 rabbit polyclonal antibody (abcam, Cambridge, MA, USA), CD (cluster of differentiation) 3 rabbit polyclonal antibody (my biosource Inc., CA, USA), CD45 rat monoclonal antibody (my biosource Inc., CA, USA), RORγt (related orphan receptor gamma t) American hamster monoclonal antibody (BioLegend, CA, USA), and γδ TCR (T cell receptor) mouse monoclonal antibody (Alexa Fluor 546 is added as a fluorescent label) (Santa Cruz Biotechnology, TX, USA) were used as the primary antibodies. Secondary antibodies were Alexa Fluor 546 Donkey Anti-Rabbit IgG (Thermo Fisher Scientific, US) to detect ADAMTS-5 and CD45, Alexa Fluor 647 Goat Anti-Rat IgG (Thermo Fisher Scientific, US) to detect CD3, and Alexa Fluor 488 goat Anti-Rabbit IgG (BioLegend, CA, USA) to detect RORγt. Nuclear staining was performed using stain solution Hoechst 33342 (Thermo Fisher Scientific, US). The number of cells was measured using the ImageJ software.

Cells were fixed in 3.5% formaldehyde for 30 min at room temperature (RT). Following this, the cells were permeabilized with 0.1% Triton X-100 for 10 min at RT. The cells were then blocked in 3% BSA for 1 h at RT. The cells were then treated with primary antibodies (1:100) overnight at 4° C. Immunofluorescent staining of primary antibodies was achieved by staining with Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 488 goat anti-mouse IgG, or Alexa Fluor 594 rabbit anti-goat IgG secondary antibodies, as appropriate (BioLegend, San Diego, CA, USA). The stained images were visualized using an FV500 confocal microscope (Olympus, Tokyo, Japan).

Briefly, primary HSCs were grown on a coverslip and fixed in a 4% paraformaldehyde solution, followed by permeabilization with 0.1% Triton X-100. The cell samples were immunostained with antibodies directed against E6AP overnight, followed by incubation with Alexa Fluor^® 488 goat anti-rabbit IgG (Invitrogen). Tissue sections were deparaffinized and incubated with antibodies of E6AP and desmin overnight and 4 h at 37 °C, respectively, followed by incubation with Alexa Fluor^® 594 goat anti-rabbit IgG (Biolegend, San Diego, CA, USA) or Alexa Fluor^® 488 goat anti-rabbit IgG (Biolegend) at 37 °C for 3 h. After incubation, the samples were cover-slipped with mounting media. The samples were examined using a laser-scanning confocal microscope (A1, Nikon instruments Inc., NY, USA).