[IF=4] Walter et al. Reduced tissue osmolarity increases TRPV4 expression and pro-inflammatory cytokines in intervertebral disc cells. (2016) Eur.Cell.Mater. 32:123-36  IHC,WB ;  Bovine, Human.  

PubMed:27434269

[IF=3.54] Sakakibara A, Sakakibara S, Kusumoto J, Takeda D, Hasegawa T, Akashi M, et al. (2017) Upregulated Expression of Transient Receptor Potential Cation Channel Subfamily V Receptors in Mucosae of Patients with Oral Squamous Cell Carcinoma and Patients with a History of Alcohol Consumption or Smoking. PLoS ONE 12(1): e0169723.  IHC-P ;  Human.  

PubMed:28081185

Paraformaldehyde-fixed, paraffin embedded (Human brain glioma); Antigen retrieval by microwave in sodium citrate buffer (pH6.0) ; Block endogenous peroxidase by 3% hydrogen peroxide for 30 minutes; Blocking buffer (3% BSA) at RT for 30min; Antibody incubation with (TRP12) Polyclonal Antibody, Unconjugated (bs-6425R) at 1:400 overnight at 4°C, followed by conjugation to the secondary antibody (labeled with HRP)and DAB staining.

NIH/3T3 cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, C-0005) at 37°C for 20 min; Antibody incubation with (TRP12/TRPV4) polyclonal Antibody, Unconjugated (bs-6425R) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.

Blank control: Raji.
Primary Antibody (green line): Rabbit Anti-TRP12 antibody (bs-6425R)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-PE
Dilution: 1μg /test.
Protocol
The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with PBST for 20 min at room temperature. The cells were then incubated in 5%BSA to block non-specific protein-protein interactions for 30 min at at room temperature .Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.

Black line : Positive blank control (Raji); Negative blank control (HL60）
Green line : Primary Antibody (Rabbit Anti-TRP12 antibody (bs-6425R) )
Orange line：Isotype Control Antibody (Rabbit IgG) .
Blue line : Secondary Antibody (Goat anti-rabbit IgG-PE)/(Goat anti-rabbit IgG-AF647)
Raji（Positive）and HL60（Negative control）cells (black) were fixed with 4% PFA for 10min at room temperature, permeabilized with PBST for 20 min at room temperature, and incubated in 5% BSA blocking buffer for 30 min at room temperature. Cells were then stained with TRP12 Antibody(bs-6425R)at 1:50 dilution in blocking buffer and incubated for 30 min at room temperature, washed twice with 2% BSA in PBS, followed by secondary antibody(blue) incubation for 40 min at room temperature. Acquisitions of 20,000 events were performed. Cells stained with primary antibody (green), and isotype control (orange).