Fitc labeled goat anti rabbit igg

Paraffin sections from testes of different development stages were prepared with the same procedure as immunohistochemistry. Sections were dewaxed and dehydrated by gradient ethanol, and then repaired by adding EDTA antigen repair buffer with microwave heating. After being blocked with 5% bovine serum albumin (BSA; Solarbio, Beijing, China), sections were incubated using anti-BCAS2 antibody (1:500 dilution; Catalog No. DF3855, RRID: AB_2836212; Affbiotech, Cincinnati, OH, USA) at 4 °C overnight. Subsequently, sections were incubated using fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG (1:200; Servicebio, Wuhan, China), and the nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI; Servicebio, Wuhan, China). Finally, sections were sealed utilizing solution containing anti-fluorescence quencher, and then observed and photographed under fluorescence microscope. Representative images were captured by CaseViewer software (3DHISTECH, Budapest, Hungary).

Double immunofluorescence labeling was performed to determine the expression patterns of GFAP-BDNF and GFAP-TrkB complexes in colon. Tissue was fixed and dehydrated as described above. After being embedded using BMJ-III, samples were sliced by a rotary slicer (Leica-2016, Germany). Slices were immersed in 0.01 M citrate buffer (pH 6.0), heated in a microwave oven at medium-high heat until boiling with an interval of 5 min. Samples were first rinsed with PBS and then blocked with 10% serum (Zhejiang Tianhang Biotechnology Co., Ltd., China) at room temperature for 30 min. Primary antibodies (anti-GFAP, mouse monoclonal antibody, concentration: 1 : 100, Abcam, UK; anti-TrkB, Rabbit polyclonal antibody, 1 : 100, Affinity Biosciences, OH, USA; anti-BDNF, rabbit polyclonal antibody, 1 : 100, Bioss, China) were added to the samples separately. After rinsing the samples with PBS for 30 min at 37°C, a secondary antibody (FITC labeled goat anti-rabbit IgG, ServiceBio, China; Cy3-labeled goat anti-mouse IgG, ServiceBio, China) was added. The samples were dripped with nuclear dye, washed with PBS, and sealed with an antifluorescence attenuation sealing agent. A fluorescence scanning microscope camera system (Jinan Tangier Electronics Co., LTD, China) was used for image acquisition.

Brain tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into coronal slices of 5 μm. Repair antigens were performed following deparaffinization and rehydration. The sections were then permeabilized using 0.3% Triton X‐100 and washed with PBS 3 times. After blocking with 5% bovine serum albumin (BSA), the sections were incubated with mouse anti‐c‐Fos (1:200, Proteintech) and rabbit anti‐CCK (1:100, Ablepsience, Henan, China) at 4°C overnight. Cy3‐labeled goat anti‐mouse IgG and FITC‐labeled goat anti‐rabbit IgG (both 1:200, Servicebio) were used as secondary antibodies. The sections were washed in PBS 3 times and then stained with DAPI (Servicebio) to reveal nuclei. At least 3 representative, non‐overlapping images at 400× magnification were obtained with a fluorescence microscope (Olympus BX51). The immunoreactivity levels were determined by analyzing the positive stained area with Image J software.