Alexa fluor 647 conjugated anti rabbit igg secondary antibody

For the staining of cell surface TRPV1, the paraformaldehyde-fixed RGCs were stained with rabbit anti-TRPV1 primary antibody (1:100, LifeSpan BioSciences) for 1 h and Alexa Fluor 647-conjugated anti-rabbit IgG secondary antibody (1:300, Thermo Fisher Scientific) for 1 h. The samples were then permeabilized using 0.3% Triton X-100 for 10 min after 4 washes in PBS. After additional 3 washes in PBS, the samples were further stained with rabbit anti-TRPV1 primary antibody for 1 h and Alexa Fluor 488-conjugated anti-rabbit IgG secondary antibody (1:500, Thermo Fisher Scientific) for 1 h to detect intracellular TRPV1. Nuclei were stained with DAPI (1:5000, Sigma-Aldrich), and the stained samples were examined by laser scanning microscopy using a LSM 880 microscope (ZEISS). The fluorescence intensity was quantified using ImageJ software.

C. difficile and L. lactis cultures were harvested by centrifugation, washed with PBS and resuspended in 4% (v/v) formaldehyde (ThermoFisher, USA) diluted in H₂O for 30 minutes at RT. After two washes with PBS, bacterial pellets were incubated with rabbit anti-CD2831 serum diluted 1:100 for 1 hour at RT. Following PBS washes, bacteria were incubated with Alexa-Fluor 647-conjugated anti-rabbit IgG secondary antibody (ThermoFisher, USA) diluted 1:500 for 30 minutes at RT. All washing steps and antibodies dilutions were performed using 1% (w/v) bovine serum albumin (BSA) in PBS. Bacterial samples were analyzed by BD FACS Canto II system (BD Bioscience, USA).

The frozen sections were washed with PBS, 0.3% Triton X-100 in PBS (15 min) and 50 mM glycine (15 min) followed by blocking with a mixture of 3% bovine serum albumin (Wako, Osaka, Japan) in PBS for 1 h at room temperature. Next, the sections were incubated with anti-DAT antibody (1:500; Merck Millipore)　or anti-GR (1:500; Santa Cruz Biotechnology, CA, USA) overnight at 4 °C. The sections were then treated with Alexa Fluor 488-conjugated anti-rat IgG secondary antibody (1:1,000; Thermo Fisher Scientific), Alexa Fluor 594-conjugated anti-rabbit IgG secondary antibody (1:1,000; Thermo Fisher Scientific) or Alexa Fluor 647-conjugated anti-rabbit IgG secondary antibody (1:1,000; Thermo Fisher Scientific), and DAPI (1:1000; Dojindo, Kumamoto, Japan) for 1 h at room temperature. After washing in 1 × PBS, sections were placed on slides, air dried, and cover slipped with Vectashield (Vector Labs, CA, USA). All fluorescently stained sections were examined with a confocal laser microscope (TiEA1R; Nikon Instech, Tokyo, Japan) and viewed using NIS-Elements software (Nikon Instech).