Zf 0312

The HUVECs were seeded into 24-well plates at a density of ~10⁴ cells per well and cultured for two days in ECM. Next, the cells were washed and fixed in 4% paraformaldehyde at 4°C for 15 min, washed with 1× PBS, blocked with 5% bovine serum albumin for 30 min and then incubated with the primary antibody at room temperature for 60 min. The expression of CD31, CD146, and GRP-78 was detected using monoclonal rabbit anti-human antibodies (dilution, 1:100; ab180175, ab75769 and ab108615, respectively; Abcam, Cambridge, UK) CD105 was detected using a monoclonal mouse anti-human antibody (dilution, 1:100; ab11414, Abcam). The negative control samples were concurrently incubated with 1× PBS. The secondary antibodies were fluorescein isothiocyanate-conjugated goat anti-rabbit and goat anti-mouse (dilution, 1:50; ZF-0311 and ZF-0312, respectively; ZSGB-BIO, Beijing, China). The nuclei were stained using DAPI (dilution, 1:1,000). The samples were viewed under an Olympus BX51 fluorescence microscope (Olympus, Tokyo, Japan) and analyzed using Image-Pro Plus 6.0 software (IPP 6.0; Media Cybernetics Inc., Rockville, MD, USA).

To investigate the spatial distribution of active Cre recombinase, tdTomato signals in various tissues from 5-month-old Lcn9-Cre; Rosa26^tdTomato mice were examined with a fluorescence stereomicroscope (M165; Leica). To determine changes over time, tdTomato signals in Lcn9-Cre; Rosa26^tdTomato mice of different ages were detected.
To visualize the precise distribution of active Cre recombinase and track the cell types with positive signals, epididymal samples isolated from the heterozygous Lcn9-Cre; Rosa26^tdTomato males were embedded in Tissue-Tek OCT (4583; Sakura). Six-μm thick frozen sections were washed with phosphate-buffered saline (PBS), blocked with 10% goat serum and incubated with primary antibodies overnight at 4°C. Immunofluorescence signals were screened after FITC-conjugated secondary antibody incubation and counterstaining with DAPI. Primary antibodies included cytokeratin 5 (Krt5) (ab52635; rabbit; Abcam; 1:200) [36 (link)] for basal cells, α-SMA (BM0002; mouse; Boster-bio; 1:200) for smooth muscle cells, and vacuolar H⁺-ATPase (V-ATPase) (ab200839; rabbit; Abcam; 1:200) [37 (link)] for narrow cells and clear cells. FITC-conjugated goat anti-mouse IgG (ZF-0312; ZSGB-bio; 1:100) and goat anti-rabbit IgG (ZF-0311; ZSGB-bio; 1:100) were used as secondary antibodies. Fluorescent images were captured with a confocal microscope (LSM900; ZEISS).

After IVIS imaging, 22Rv1 tumors were dissected and embedded in an optimal cutting temperature compound (OCT). Then the tumor tissues were cut into 20 μm thick slides and fixed with acetone at 4 °C for 20 min. After being blocked by BSA at room temperature for 1 h, the slides were incubated with a primary antibody against PSMA (ab19071; clone YPSMA-1, Abcam, Cambridge, UK; 1:300 dilution) at 4 °C overnight, and then stained with a FITC-labeled secondary antibody (ZF-0312; ZSGB-Bio, Beijing, China; 1:150 dilution) at room temperature for 1 h. Finally, DAPI was used for counterstaining. Vectra Polaris was used to scan the stained slides.

A step-by-step protocol of immunofluorescence staining can be found at Protocol Exchange⁵⁷ . Fresh resected specimens were embedded in optimal cutting temperature compound (OCT) and stored at −80 °C until for immunofluorescence. The embedded specimens were cut into 7 μm-thick slides and fixed with cold acetone for 20 min. After being blocked by BSA for 1 h, the slides were incubated with a primary antibody against PSMA (ab19071; Abcam, Cambridge, UK; 1:1000 dilution) at 4 °C overnight, and then stained with FITC-labeled secondary antibody (ZF-0312; ZSGB-Bio, Beijing, China; 1:100 dilution) at room temperature for 1 h. Finally, Hochest 33342 was used to stain nuclei and a 10 μM ODAP-490 solution was applied before the slides were sealed. The FluoView1000 confocal microscope (Olympus, Japan) was used to scan the stained slides.