Donkey anti goat alexa488

Coverslips with plated neurons were fixed with 4% PFA and blocked with superblock (Thermo Fisher Scientific) for 30 min. Human HNSCC tissues were resected and fixed with 4% PFA, dehydrated, embedded in paraffin, and cut into 8-μm sections. Sections were then deparaffinized and blocked with superblock. H&E staining was performed to confirm cancer lesions. For immunofluorescence labeling, neurons or tissue sections were then incubated for 24 h at 4°C in rabbit anti-P2X3 (1:500, Alomone Labs) and goat anti-P2X2 (1:500, Santa Cruz Biotechnology). The sections were then washed in phosphate-buffered saline (PBS) with Triton X-100 and incubated in secondary antibody chicken anti-rabbit Alexa-594 (1:1000, Invitrogen) and donkey anti-goat Alexa-488 (1:1000, Invitrogen) in a dark chamber for 2 hours at room temperature. Control experiments were performed by incubation in secondary antibody alone and by applying P2X2 blocking peptides (Santa Cruz Biotechnology), and P2X3 blocking peptides (Alomone Labs). The coverslips or sections were washed and visualized with images acquired using a Nikon Ti Eclipse microscope (Nikon).

The number and type of inflammatory cells were characterized in the prostate of Akita and WT mice. Three random areas from each prostatic lobe from each animal were acquired (WT: n = 4; diabetic: n = 4). Neutrophils were identified and quantitated in H&E stained sections. Immunohistochemistry was used to quantitate monocytes/macrophages, T lymphocytes, B lymphocytes, and fibrocytes. Briefly, sections were blocked for 4 hours in PBS containing 10% donkey serum and 1% BSA (both from Sigma-Aldrich, St. Louis, Missouri) was followed by primary antibodies-rat anti-F4/80 (1 : 50, eBioscience, San Diego, CA), rabbit anti-CD3 (1 : 100, Dako, Carpinteria, CA) and goat anti-CD20 (1 : 100, Santa Cruz, Santa Cruz, CA), and rat anti-CD45 (1 : 100, Abcam, Cambridge, MA) and rabbit anti-vimentin (1 : 100, Abcam, Cambridge, MA) overnight at 4 degrees. Secondary antibodies-donkey anti-rat Alexa 594, donkey anti-rabbit Alexa 594, donkey anti-rat Alexa 488, and donkey anti-goat Alexa 488 (1 : 100, Invitrogen, Grand Island, NY) were incubated for one hour at room temperature. Four μg/mL of Hoechst 33258 (Sigma-Aldrich, St. Louis, Missouri) was incubated for 10 minutes. For quantification of each cell type, three random areas from each prostatic lobe from each animal were acquired using Nikon eclipse Ti-U microscope.

Immunofluorescence of H-PGDS, c-kit and COX-2 was performed, as described previously^{29 (link)}. Briefly, excised colons were fixed in 4% paraformaldehyde at 4 °C for 2 h. Cryosections (4 μm) were blocked with PBS containing 0.05% Triton X-100 and 3% normal goat serum albumin at room temperature for 15 min. The sections were then immunostained using primary antibodies against H-PGDS (Cayman Chemicals, 1:500, rabbit), c-kit (Merck Chemical, Darmstadt, Germany, 1:125, rat) and COX-2 (Santa Cruz Biotechnology, Santa Cruz, CA, 1:100, goat) at 4 °C overnight. The secondary antibodies, goat anti-rabbit Alexa 568 and goat anti-rat Alexa 488 or donkey anti-goat Alexa 488 (Invitrogen, Carlsbad, CA, 1:500), were incubated at room temperature for 2 h. Images were captured using an Eclipse E800 fluorescence microscope (Nikon, Tokyo, Japan).

The following primary antibodies were used: goat polyclonal anti‐VE‐cadherin (1:200, sc‐6458, Santa Cruz Biotechnology), anti‐β‐catenin (1:100, #610 154, BD Transduction Laboratories), rabbit polyclonal anti‐pY658‐VEC^[14] (1µg mL⁻¹), and mouse monoclonal anti‐58K Golgi protein (1:100, ab27043, Abcam). The secondary antibodies were donkey anti‐goat‐Alexa 488 (Invitrogen, A‐11055), donkey anti‐Mouse‐Alexa 555 (Invitrogen, A‐31570), chicken anti‐rabbit‐Alexa 647 (Invitrogen, A‐21443), and donkey anti‐mouse‐Alexa 488 (Invitrogen, #A21202).

Embryos were washed twice with DPBS supplemented with 0.1% BSA and fixed with 4% paraformaldehyde in DPBS at room temperature (RT) for 15 minutes. Fixed embryos were permeabilized using 0.2% Tween-20 and 0.2% Triton X-100 in DPBS at RT for 15 minutes, followed by blocking with 10% donkey serum in DPBS at RT for 1 hour. Samples were stained with anti-SOX2 (5 μg/mL) and anti-SOX17 (1 μg/mL) in DPBS containing 10% donkey serum at 4°C overnight. After washing 3 times in washing solution (DPBS with 0.2% Tween-20 and 1% BSA for 10 minutes), embryos were incubated with donkey anti-rabbit Alexa594 or donkey anti-goat Alexa488 (Invitrogen, Waltham, MA, USA; 1:5,000) in DPBS with 10% donkey serum at RT for 1 hour. For double staining, samples were stained again with primary and secondary antibodies. The steps were the same, but primary antibody treatment was conducted at RT for 2 hours. All samples were washed 3 times with washing solution after secondary antibody treatment. Immunostained embryos were mounted on a glass slide with Prolong Gold and 4′, 6-diamidino-2-phenylindole (DAPI) (Invitrogen, USA) and cured for more than 24 hours. We described the list of antibodies in Table 3. The digital imaging system for microscope (DS-L1; Nikon, Japan) was used to obtain fluorescence and bright-field images. We used the ImageJ program for images.