Alexa fluor 488 conjugated goat anti mouse secondary antibody

Fixed low-passage poorly-differentiated human MEC cells from 2D and 3D culture were incubated overnight at 4 °C with mouse monoclonal antibodies against HIF1α (1:200; Abcam). After rinsing in PBS, cells were further incubated with Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (1:500; Abcam) and rinsed. Negative controls included omission of primary or secondary antibodies and substitution of primary antibodies with isotype controls. After mounting in anti-fading mounting medium (Beyotime, Beijing, China), stained cells were visualized and imaged with a fully motorized, Olympus IX83 inverted wide-field epifluorescence microscope with a DP80 CCD camera under the monochromatic mode controlled by Olympus cellSens Dimension software (Olympus; Tokyo, Japan).

The immunocytochemical assay was conducted as previously described [28 (link)] with some modification. Briefly, hemocytes collected from healthy shrimp were suspended in the 5% BSA containing resuspending solution [29 (link)], seeded in a cell culture dish (NEST, Wuxi, China) for 3 h, and incubated with rMBP-HSSP-His (50 μg/mL) at room temperature. The same concentration of rMBP-His was used as the negative control. The hemocytes were fixed on dishes with 4% paraformaldehyde prepared in PBS (pH 7.4) for 15 min at room temperature, and washed three times in TBS (50 mM, Tris–HCl, 150 mM NaCl, pH 7.4). After blocking in 5% BSA in TBS at 4 °C overnight, the hemocytes were incubated with mouse anti-MBP antibody (ABclonal, Wuhan, China) in a dilution of 1:500 at room temperature for 1 h, and then incubated with Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (Abcam, Shanghai, China) at a dilution of 1:1000 at room temperature for 1 h. Hemocytes were dyed with Di1 (Beyotime, Shanghai, China) for 20 min and DAPI (Beyotime, Shanghai, China) for 5 min before the observation under a laser confocal scanning microscopy (Carl Zeiss LSM 710, Carl Zeiss, Oberkochen, Germany).

The full-length E165R gene was amplified by PCR using the synthesized construct as the template (Table S1). The PCR product was cloned into pCAGGS-HA vector (Addgene, Teddington, UK) using EcoR I and Xho I restriction sites. PK15 cells (porcine kidney cell) were transfected with 500 ng of plasmid per well in a 24 well plate. The culture medium was replaced with DMEM supplemented with 10% FBS at 6 h post transfection. The cells were fixed with 3% paraformaldehyde for 30 min before being washed 3 times with PBS and then incubated with anti-E165R mAb (1:500) or anti-HA tag mAb (NO: ab18181; Abcam, Cambridge, UK) (1:1000). After 1 h of incubation at 37 °C, cells were washed 3 times with PBS and subsequently incubated with Alexa Fluor-488-conjugated goat anti-mouse secondary antibody (NO: ab150113; Abcam, Cambridge, UK) (1:500) at 37 °C for 1 h. Following the washing step 3 times with PBS, the cells were observed under the OLYMPUSIX IX73 plore Standard fluorescence microscope (OLYMPUSIX, Tokyo, Japan).

Following treatment for five days, cells in transparent collagen IV-coated plates were washed twice with phosphate-buffered saline (PBS) at room temperature and fixed for 5 min using absolute methanol (precooled to -20 °C). Cells were washed thrice with ice-cold PBS and incubated with blocking buffer PBST (1% (w/v) bovine serum albumin/10% (v/v) normal goat serum/0.3 M glycine in PBS containing 0.1% (v/v) Tween-20) for 1 h. Cell body and processes were then labeled with an anti-βIII-tubulin mouse primary antibody (1 µg/mL; Abcam, Cambridge, UK) diluted in PBST containing 1% (w/v) bovine serum albumin at 4 °C overnight, followed by an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (2 µg/mL; Abcam, Cambridge, UK) for 1 h at room temperature, protected from light. For nuclear staining, cells were counterstained with Cellstain^® 4′,6′-Diamidino-2-phenylindole (DAPI; Dojindo Molecular Technologies, Kumamoto, Japan) for 10 min at 37 °C. Plates were then subjected to image acquisition and analysis of neurite outgrowth.

Specimens cultured in osteogenic medium for 7, 14 and 21 days were fixed with 2% (w/v) paraformaldehyde (Nacalai Tesque Inc., Kyoto, Japan) for 15 min. They were incubated with PBS, containing 0.25% Triton X-100 (Sigma) (PBS-T) for 10 min for permeabilization, and then incubated in PBS-T with 1% (w/v) bovine serum albumin (Sigma) for 30 min to block non-specific binding [16 (link)]. Specimens were incubated with a rat anti-osteocalcin (OCN) antibody (1:200 dilution, Takara Bio Inc., Kusatsu, Japan) and rabbit anti-osteopontin (OPN) antibody (1:1000 dilution, Abcam, Cambridge, MA, USA) for 1 h, followed by an incubation with an Alexa Fluor^® 488-conjugated goat anti-mouse secondary antibody (1:1000 dilution, Abcam) and Alexa Fluor^® 555-conjugated goat anti-rabbit secondary antibody (1:1000 dilution, Abcam) for 1 h. Specimens were also incubated with 4′,6-diamidino-2-phenylinole (DAPI) (Invitrogen^TM/Life Technologies) diluted in PBS for 5 min to identify nuclei. Immunofluorescence images were evaluated using immunofluorescence microscopy (Leica M165FC, Leica Microsystems, Nussloch, Germany).