Alexa fluor 488 conjugated donkey anti mouse

HeLa cells were grown on coverslips for twenty-four hours and were washed twice
with PBS and fixed with 4% paraformaldehyde in PBS (pH-7.4) for
10 min. After washing with PBS, cells were quenched with
30 mM glycine. Cells were then permeabilized with 0.1% Triton-X-100
for 10 min and blocked with 10% normal goat serum (NGS) in PBS for
1 h. Cells were incubated with appropriate antibodies (anti-Calnexin;
1:200 from SCB, anti-Giantin; 1:1000 from Abcam) for 1 h in PBS
containing 2% NGS. The cells were washed three times with PBST and incubated
with Alexa-Fluor 488-conjugated donkey anti-mouse and Alexa-Fluor 568-conjugated
donkey anti-rabbit (Life Technologies) antibodies for 30 min. The
cells were washed three times with PBST and mounted in Vectashield with DAPI
(Vector Laboratories).

CAFs and NFs were collected and seeded into 6-well plates coated with polylysine. Following three rinses with preheated 0.01 mol/L PBS, the cells were fixed with 4% paraformaldehyde under room temperature conditions for a 30-min period. After three PBS rinses, the cells were sealed with blocking solution (Beyotime Biotechnology Co., Shanghai, China) at 37 °C for 60 min. Afterwards, the cells were probed with rabbit antibodies against α-smooth muscle actin (α-SMA; ab32575, 1: 200), fibroblast activation protein (FAP; ab53066, 1: 50), and fibroblast-specific protein 1 (FSP1; ab124805, 1: 500) overnight at 4 °C. The antibodies were obtained from Abcam Inc., Cambridge, UK. The following day, further incubation of the cells was conducted with the following secondary antibodies: Alexa Fluor 594 conjugated donkey anti-rabbit (1: 400, A21202) and Alexa Fluor 488 conjugated donkey anti-mouse (1: 400, A21207), for 1 h in dark, which were both purchased from Life Technologies (Carlsbad, CA, USA). Subsequently, the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime Biotechnology Co., Shanghai, China) at room temperature for 5 min, and sealed with anti-fade mounting medium (Beyotime Biotechnology Co., Shanghai, China). Finally, a high-content screening imaging system (Image Xpress Micro 4, Molecular Devices, Sunnyvale, CA, USA) was employed for photography.

HeLa cells grown on cover slips were transiently transfected with pSecTag2C-HA-MUC16-114-Myc (i.e. HA114Myc) plasmids using Lipofectamine (Invitrogen). HeLa cells grown on cover slips were transiently transfected with p3X-FLAG-CMV9 (CMV9), p3X-FLAG-CMV9-114HA (CMV9-F114HA), p3X-FLAG-CMV10 (CMV10) and p3X-FLAG-CMV10-114HA (CMV10-F114HA) using Lipofectamine. MiaPaCa-2 and T3M4 cells stably transfected with p3X-FLAG-CMV9 and p3X-FLAG-114HA were grown on cover slips for 24 hours. Then the cells were washed twice with PBS and fixed with 4% paraformaldehyde in PBS (pH-7.4) for 10 min. After washing with PBS, cells were quenched with 30 mM glycine. Cells were then either non-permeabilized or permeabilized with 0.1% Triton-X-100 for 10 min and blocked with 10% normal goat serum (NGS) in PBS for 1 h. Cells were incubated with appropriate antibodies (anti-HA; 1:300, anti-FLAG; 1:500, anti-Myc; 1:300, anti-JAK2; 1:300) for 1 h in PBS containing 2% NGS. Cells were washed with PBST (x3) and PBS (x1) and incubated with Alexa Fluor 488-conjugated donkey anti-mouse and Alexa Fluor 568-conjugated donkey anti-rabbit (Life Technologies) antibodies for 30 min. The cells were washed with PBST (x3) and PBS (x1) and mounted in Vectashield with DAPI (Vector Laboratories).

Intact embryos were fixed in 2.5%PFA/ethanol [95 (link)] or methanol/acetone [96 (link)] for all immunofluorescence except for those probed with monoclonal antibody H5, which was fixed in methanol/formaldehyde [95 (link)]. Primary antibodies used were: anti-H3K4me2 (CMA30) 1:1000 END Millipore], anti-P-granules [OIC1D4, 1:5 [96 (link), 97 (link)])], anti-H3K36me3 [(CMA333), 1:1000 [95 (link)]], anti-Ser2p RNA pol II CTD (H5, 1:500, Covance MMS-129R), anti-GFP (1:1000, Novus NB600-308), anti-AMA-1 (1:10,000, Novus 38520002), and anti-FLAG (M2, 1:1000, Sigma F1804). Secondary antibodies used were Alexa Fluor 488-conjugated donkey anti-mouse (1:500, Invitrogen R37114) and Alexa Fluor 594-conjugated goat anti-rabbit (1:500, Invitrogen R37117). Samples were mounted in ProLong Gold anti-fade reagent (Life technologies, P36934) and observed under a fluorescence microscope (Leica DMRXA; Hamamatsu Photonics, Hamamatsu, Japan) with Simple PCI software (Hamamatsu Photonics). Image J was used for quantification of raw immunofluorescence intensity [98 ].

VCR was ordered from Selleck Chemicals (Houston, TX, United States). OT, carmine red, and carboxymethylcellulose were ordered from Sigma-Aldrich Corp (St Louis, MO, United States). Isoflurane was ordered from RWD (Shenzhen, China). VCR and OT were dissolved and attenuated in normal saline (NS). carmine red (6%) was suspended in 0.5% carboxymethylcellulose. Primary antibodies against NeuN, β Ⅲ Tubulin, nNOS, choline acetyltransferase (ChAT), Nrf2, p44/42 MAPK, phospho-p44/42 MAPK, p38 MAPK, phospho-p38 MAPK, and GAPDH were ordered from Gene Tex (Irvine, United States), Cell Signaling Technology (Danvers, MA, United States), Abcam (Cambridge, UK), and Proteintech (Chicago, United States). Fluorescent secondary antibodies of Alexa Fluor 568-conjugated donkey anti-rabbit and Alexa Fluor 488-conjugated donkey anti-mouse were ordered from Invitrogen Life Technology (Foster City, CA, United States). DAPI was ordered from Beyotime Biotechnology (Shanghai, China). Dihydroethidium (DHE) was ordered from Sigma-Aldrich Corp. HRP-conjugated goat anti-rabbit secondary antibody was ordered from Zhongshan Golden Bridge Biotechnology (Beijing, China). Stripping buffer was ordered from CWBIO (Taizhou, China).