Levels of active caspase-3 were also determined using a specific rabbit anti-active caspase-3 antibody (R&D Systems).
Cryosections (5 μm thick) of tumors pre-infected with 2×10 cfu (colony forming units) were fixed with 4%% paraformaldehyde (PFA), permeabilized with 0.2 % Tween 20 in PBS and blocked with 1 % BSA in PBS prior to staining using a specific rabbit anti- Salmonella lipopolysaccharide (LPS) O Poly A antiserum (BD Biosciences).
The membrane was blocked in a 5% milk TNT buffer (Tris-HCl, pH 7.4, 140 mM NaCl, and 0.05% Tween 20) overnight, and then probed using a specific rabbit polyclonal anti-Orai1 antibody (1 : 200, ProScience, Poway, CA, USA) anti-mouse monoclonal ant-STIM1 antibody (1 : 250, BD Biosciences, Mountain View, CA, USA) and anti- β-actin (Lab Vision Co., Fremont, CA, USA, 1/1000) antibodies.
10 We used a specific rabbit polyclonal antibody against Cyr61 (H-78, Santa Cruz Biotechnology, Santa Cruz, CA) and mouse monoclonal antibodies to CD204 (SRA-E5, Trans Genic, Kobe, Japan), CD163 (10D6, Novocastra, Newcastle upon Tyne, UK), and CD68 (Kp-1, DAKO) for the primary reaction.
Non-specific antibody binding was blocked using a specific blocking reagent for rabbit primary antibodies (Sniper, Biocare Medical, Walnut, Creek, CA, USA) for 5 min.
Two rabbits were immunized using a specific synthetic peptide for each protein: AtJ8 111-1344) (NH2-) CKNQMEGTEEFEPFDVYDEGLNGMN (-CONH2); AtJ11 (127-136) (NH2-) CSVYDRRMLRR (-CONH2); AtJ20 (145-158) (NH2-) CRQNRYDQEVVEEKS (-CONH2).
Plasma LH level was assayed using a highly specific rabbit anti-serum, anti-native rat LH (ALPCO diagnostics # 29-R002) wI125I125 as tracer.
Upon heterologous expression in HEK-293 cells, AC8 can be detected by immunoblotting in crude membranes using a highly specific rabbit polyclonal antibody raised against the COOH terminus of AC8 (3) (Fig. 1).
Placentas from the 32 PCR-positive women and 10 PCR-negative controls were tested for W. chondrophila by using immunohistochemical analysis with a specific rabbit polyclonal antibody as described (10 ).
Urinary aMT6s was assayed using 96 well ELISA kits (Bühlmann Labs, EK-M6S) purchased from ALPCO, Ltd. (Windham, NH), a competitive immunoassay that uses a highly specific rabbit anti-6-sulfatoxymelatonin antibody and a second antibody capture technique.
Immunohistochemistry for cleaved caspase-3 was performed with the Vector Elite ABC-HPHP kit (Vector, Burlingame, CA, USA) using a murine-specific rabbit anti-active capase-3 (BD Pharmingen, San Jose, CA, USA) for detection, and goat anti-rabbit biotinylated antibody (Vector) for labeling, as described previously [ 17].
