Alexa fluor plus 555

Immunofluorescence staining was used to analyze 4% paraformaldehyde-fixed, 0.1% Triton X-100-permeabilized WT MCA205 and Six1^−/− MCA205 cells labeled with an anti-collagen VI antibody (1:200, Abcam, cat# ab182744) at 4 °C overnight. After washing three times, the cells were incubated with an Alexa Fluor Plus 555-conjugated (1:500, Invitrogen, A32732) secondary antibody for 1 h. Cell nuclei were counterstained with Hoechst 33258 (Thermo Fisher Scientific, H3569) for 5 min at RT. Images were acquired using a confocal microscope (Leica TCS SP8).
Tumors were harvested on day 8 or 12, fixed in 4% paraformaldehyde for 24 h, dehydrated in a 30% (wt/vol) sucrose solution for 48 h and finally embedded in OCT at room temperature. Four-micrometer frozen sections of tumor tissues were obtained and fixed in ice-cold acetone for 15 min. After blocking with 5% goat serum in PBS for 1 h, the tumor tissue sections were incubated with primary antibodies against CD8a (1:100, Abcam, ab217344) at 4 °C overnight. After washing three times, the tumor tissue sections were incubated with an Alexa Fluor Plus 555-conjugated (1:500, Invitrogen, A32732) secondary antibody for 1 h. Cell nuclei were counterstained with Hoechst 33258 (Thermo Fisher Scientific, H3569) for 5 min at RT. Images were acquired using a confocal microscope (Leica TCS SP8) and analyzed with ImageJ software.

Lunaphore MK03 imaging chips have been used with the automated staining and imaging platform COMET™ for all immunostaining experiments. Generic reagents used during the staining and washing steps are as follows: De-ionized water, Alcohol 70%, Multistaining Staining Buffer (Lunaphore BU06), Elution Buffer (Lunaphore BU07-L), Quenching Buffer (Lunaphore BU08-L), Imaging Buffer (Lunaphore BU09), Blocking Buffer (Lunaphore BU10).
Primary antibodies, along with their corresponding protocol conditions, including concentration and incubation time, are listed in Supplementary Table 1.The used secondary antibodies and counterstaining solution are as follows: Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 555, Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 647, Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 555, Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 647, Goat anti-Chicken IgY (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 555, Goat anti-Rat IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 647 (Thermo Fisher Scientific), DAPI Solution (Thermo Fisher Scientific).

The dissected mouse cochleae or cultured HEI-OC1 cells were fixed in 4% paraformaldehyde. After washsing three times with 1% Triton X-100 in PBS (PBST) for 5 min each time, the samples were blocked with 10% donkey serum for 1 h at room temperature, followed by incubation with primary antibody at 4°C overnight. After washing three times with PBST, samples were incubated with secondary antibody with or without phalloidin for 1.5 h at room temperature, then washed three times again for 5 min each time and imaged under a confocal microscope.
The primary antibodies used in the present study were: myosin 7a (Proteus Bioscience, 25–6790, 1:1000 dilution) and cleaved caspase-3 (Cell Signaling Technology, #9661, 1:400 dilution). The secondary antibodies were: goat anti-rabbit, Alexa Fluor Plus 488 (Thermo Fisher, A32731, 1:400 dilution); goat anti-mouse, Alexa Fluor Plus 555 (Thermo Fisher, A32727, 1:400 dilution), goat anti-rabbit, Alexa Fluor Plus 555 (Thermo Fisher, A32732, 1:400 dilution), and Alexa FluorTM 594-conjugated phalloidin (Thermo Fisher, A12381, 1:400 dilution).

Slides were deparaffinized in xylene and rehydrated with decreasing concentrations of ethanol. Antigen retrieval was performed by using IHC-Tek™ Epitope Retrieval Solution (IHC World (Ellicott City, MD, USA), IW-1100-1L) in a steamer. Autofluorescence was blocked by using Image-iT FX Signal Enhancer (Invitrogen, Thermo Fisher Scientific, (Waltham, MA, USA), cat. no. I36933). The protein was blocked by using Normal Goat Serum, 2.5% (Vector Laboratories (Newark, CA, USA), S-1012), and then incubated overnight with primary antibody (rabbit anti-GFP IgG 1:1000 (Abcam (Cambridge, MA, USA), ab5665)) diluted in Antibody Diluent (Dako (Santa Clara, CA, USA), S0809). Slides were incubated the next day in diluent with goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 555 (Invitrogen, A32732) at 1:100. Autofluorescence was quenched by using Vector TrueVIEW (Vector Laboratories, cat. no. SP-8400), and nuclei were counterstained with antifade mounting medium with DAPI Hardset (Vector Laboratories, H-1200).

The following primary antibodies were used: anti-NOTCH1 (1:500, rabbit IgG, 3608, Cell Signaling Technology, Danvers, MA, USA), anti-HES1 (1:200, rabbit IgG, 11988, Cell Signaling Technology), anti-RBPJ (1:1000, rabbit IgG, 5313, Cell Signaling Technology), anti-JAG1 (1:200, rabbit IgG, ab109536, Abcam, Cambridge, UK), anti-DLL1 (1:100, mouse IgG2b, MAB18181, R&D, Minneapolis, MN, USA), anti-POU4F3 (1:200, mouse IgG1, sc-81980, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-ATOH1 (1:500, rabbit IgG, 21215-1, Proteintech, Rosemont, IL, USA), anti-SOX2 (1:200, goat IgG, AF2018, R&D), anti-MYOSIN7A (1:30, mouse IgG1, 138-1-s, DSHB, Iowa City, IA, USA), anti-MYOSIN7A (1:100, rabbit IgG, 25-6790, Proteus Biosciences, Ramona, CA, USA), anti-CDKN1B (1:200, mouse IgG1, 610242, BD, Franklin Lakes, NJ, USA). The following secondary antibodies were used: donkey anti-rabbit IgG, Alexa Fluor Plus 555 (1:500, A32794, Invitrogen), donkey anti-mouse IgG, Alexa Fluor Plus 488 (1:500, A32766, Invitrogen), and donkey anti-goat IgG, Alexa Fluor 647 (1:500, 705-605-147, Jackson Immuno-Research).

Adult pig ovaries were embedded in paraffin and cut into 5‐μm sections. IFA of ovary sections was performed using methods described previously (Li et al., 2014). Antibodies against NRDR (1:100, Abcam, CA) were added to the sections and incubated at 4°C for 12 hr. After washing, sections were incubated with the goat antirabbit IgG (H + L) highly cross‐adsorbed secondary antibody, Alexa Fluor Plus 555 (1:500; Invitrogen, CA) at room temperature for 3 hr. For the NC, sections were incubated with a rabbit IgG antibody following the same abovementioned procedures. Sections were then incubated with 4′,6‐diamidino‐2‐phenylindole (DAPI) for 10 min. Slides were viewed under a microscope (Leica Microsystems, Cambridge, UK) and photographed.

Immunohistochemical analysis was performed as previously described [25 ]. Mice were anesthetized at different time points after ICH induction, and frozen sections with the largest bleeding area were selected for immunofluorescence analysis. The following antibodies were used: mouse anti-GFAP (1:300; CST), mouse anti-Iba1 (1:250; GeneTex), mouse anti-NeuN (1:300; CST), rabbit anti-Homer1 (1:200; Abcam), rabbit anti-C3 (1:200; Abcam), rabbit anti-S100A10 (1:300; Proteintech), donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor Plus 488 (1:1000; Invitrogen), and donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor Plus 555 (1:1000; Invitrogen).