Alexa fluor 555 conjugated goat anti mouse igg h l

The middle section of the colon tissue was fixed in 10% formalin overnight and 5 μm paraffin embedded cross sections were prepared. After dewaxing and rehydration, tissue sections were boiled for 20 min in 10 mmol/L citrate buffer (pH 6.0). The sections were blocked with blocking buffer for one hour and then incubated with anti-mouse CD68 mAb (for macrophages, 1:100, Abcam Inc, Cambridge, MA, USA, ab955) or rat anti-mouse CD3 (for T cells, 1:50, Abcam Inc, Cambridge, MA, USA, ab11089) mAb separately at 4 °C overnight. After washing, the sections were incubated with Alexa Fluor^® 555 conjugated goat anti-mouse IgG (H+L) (for macrophages, 1:1000, Thermo Fisher Scientific Inc, Waltham, MA, USA, A32727) or Alexa Fluor^® 488 conjugated goat anti-rat IgG (H+L) (for T cells, 1:1000, Thermo Fisher Scientific Inc, Waltham, MA, USA, A-11006) for two hours at room temperature. The sections were then counterstained and mounted with ProLong^® Gold Antifade Mountant with DAPI (Thermo Fisher Scientific Inc, Waltham, MA, USA, P36931). Rabbit isotype control IgG (Abcam Inc, Cambridge, MA, USA, ab27478) at the same dilution was used as control. For each slide, 10 randomly chosen fields (20X) were examined and counted for fluorescent cells. The result for each slide was expressed as the average cell counts in ten fields.

Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% triton X‐100 and blocked by 1% normal goat serum, 1% BSA and 0.1% tween20 in PBS for 1 h. Those samples were then incubated at 4°C overnight with primary antibodies diluted in PBS containing 1% BSA and 0.1% tween20. Next, those samples were incubated with secondary antibodies at room temperature for 1 h. Hoechst 33342 (Doujin Chemical, Kumamoto, Japan) was used to counterstain the nuclei. The following primary and secondary antibodies were used: anti‐S100β (Abcam, Cambridge, UK, ab52642, 1:200), GFAP (Cell Signalling Technology, Beverly, MA, 3670, 1:1000), NESTIN (Millipore‐Sigma, St. Louis, MO, MAB5326, 1:500), Alexa Fluor 488‐conjugated Goat anti‐Rabbit IgG (H + L) (Thermo Fisher Scientific, A‐11034, 1:1000) and Alexa Fluor 555‐conjugated Goat anti‐Mouse IgG (H + L) (Thermo Fisher Scientific, A‐21422, 1:1000). After immunostaining steps, cells were analysed by Opera Phenix (PerkinElmer, Waltham, MA) with 10× or 40× objective lenses, followed by quantification for the positivity of astrocytic markers using Harmony software (PerkinElmer). When calculating GFAP aggregates, the images were taken using a 40× objective lens and the round areas showing strong intensity against the backgrounds were counted (Common Threshold: 0.90, Area > 10 pixels, Roundness > 0.80).

Cells were fixed with 4% PFA in PBS for 10 min at 4°C and processed for immunofluorescence analysis as previously described (Testa et al., 2017 (link)). Briefly, cells were washed with PBS and blocked with 10% goat serum in PBS for 1 h at RT. Subsequently, cells were incubated with the primary antibody anti-myosin heavy chain (MF20, mouse monoclonal, DHSB, diluted 1:2) or anti-ki67 (rabbit polyclonal, Novus Biologicals #NB110-89717, diluted 1:200) for 1 h, followed by incubation with Alexa Fluor 555-conjugated goat anti-mouse IgG (H + L; Thermo Fisher Scientific #A21422, diluted 1:400) and 488-conjugated goat anti-rabbit IgG (H + L; Thermo Fisher Scientific #A11008, diluted 1:400) for 1 h. Finally, nuclei were stained with 300 nM DAPI (Thermo Fisher Scientific) for 10 min. Specimens were viewed using a Nikon TE 2000 epifluorescence microscope equipped with a Photometrics Cool SNAP MYO CCD camera.