Alexa fluor 488 labelled goat anti mouse igg h l

Tumor tissues collected from gastric cancer patients were fixed in 4.0% paraformaldehyde, embedded in paraffin and cut into 4-μm sections. Sections were incubated with 3.0% hydrogen peroxide to inactivate endogenous peroxidase and then blocked in 5.0% bovine serum albumin after antigen retrieval. To observe the relationship between GC-MSCs and TAM location within tumor, the sections were incubated at 4 °C overnight with primary antibodies against α-SMA (NBP2–33006, Novus Biologicals, Briarwood Avenue, CO, USA) and CD204 (bs136214, absin, Shanghai, China). Subsequently, incubation with Alexa Fluor 488-labelled goat anti-mouse IgG (H + L) and Alexa Fluor 555-labelled donkey anti-rabbit IgG (H + L) (Beyotime, Shanghai, China) was performed for 1 h at 37 °C in the dark. Nuclei were counterstained with DAPI (Beyotime). Fluorescent images were acquired by a confocal laser-scanning microscope (Ti2-E-A1, Nikon, Tokyo, Japan).

Eyes obtained on day P17 were placed into 4% paraformaldehyde for 2 h. Then the retinas were sheared into flat mounts and blocked with 5% goat serum and 0.4% Triton X-100 for 1 h, followed by incubation with primary antibodies at 4 °C overnight. Then the retinas were washed carefully and incubated with secondary antibody combinations for 1 h. Images were taken by confocal microscopy (Zeiss, Germany). CellSens Dimension software was used to determine the number of microglia (Iba1⁺). Relative fluorescence intensity was measured by ImageJ. The following primary antibodies were used: CD31 (Abcam, ab9498, diluted 1:1000); IBA1 (WAKO, 019–19,741, diluted 1:1000); Pan-Kla (PTM-1401, diluted 1:200);YY1-K183la (PTM, diluted 1:200); YY1 (Proteintech, 66281–1-lg, diluted 1:200); p300 (Santa Cruz, sc48343, diluted 1:200); and Ki67 (Abcam, ab16667, diluted 1:200). The following secondary antibodies were used: Alexa Fluor 488-labelled goat anti-rabbit IgG (H + L) (Beyotime, A0423); Alexa Fluor 488-labelled goat anti-mouse IgG (H + L) (Beyotime, A0428); Cy3-labelled donkey anti-goat IgG (H + L) (Beyotime, A0502); Cy3-labelled goat anti-mouse IgG (H + L) (Beyotime, A0521); Cy3-labelled goat anti-rabbit IgG (H + L) (Beyotime, A0516); Alexa Fluor 647-labeled goat anti-mouse IgG (H + L) (Beyotime, A0473). All secondary antibodies were diluted 1:500.