Vector novared peroxidase substrate kit

Monolayers were fixed in ice-cold methanol, while pellet cultures were placed in Tissue-Tek® Cryomold® Standard (Sakura Fintek, Torrance, CA, USA) before being cryopreserved in liquid nitrogen. The frozen pellets were sectioned, placed on SuperFrost® cryosection slides (Thermo Fischer Scientific GmbH) and fixed with 3% ice-cold acetone (Carl Roth GmbH). Alizarin Red S, Oil redO and alcian blue stainings were performed as outlined previously [23 (link), 24 (link), 26 (link)]. In addition, immunohistochemical stainings were performed on monolayers and pellets using the following antibodies: Col I - monoclonal anti Col Iα1 (5 μg/mL; Abcam pls, Cambridge, Great Britain); Col II - polyclonal Col IIα1 antibodies (5 μg/mL; Acris Antibodies GmbH, Herford, Germany); Col X - polyclonal Col X antibodies (5 μg/mL; Abcam pls). The immunostainings were visualised with the Avidin-Biotion complex method using the protocols, biotinylated antibodies, blocking serum and peroxidase from the VECTASTAIN® Universal Elite® ABC Kit (Vector Laboratories, Burlingame, CA, USA) and the VECTOR® NovaRED™ peroxidase substrate kit (Vector Laboratories). The slides and wells were counterstained with haematoxylin (Sigma-Aldrich). For control stainings the primary antibodies were replaced with non-immune IgG antibodies (Sigma-Aldrich).

Cryopreserved human and mouse liver tissues were sectioned in 6 μm and fixed in cold acetone. For in vitro cell samples, cells were fixed and permeabilized using 4% paraformaldehyde and 0.5% Triton X-100. After serum blocking, samples were incubated with primary antibodies and visualized by fluorochrome-conjugated secondary antibodies. Samples were counter-stained and mounted using ProLong Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific).
For immunohistochemical staining, paraffin-embedded mouse liver samples were sectioned in 4 μm and then subjected to de-wax, rehydrate, and antigen retrieval. Endogenous peroxidase activity and biotins were quenched using hydrogen peroxide and Avidin/Biotin Blocking Kit (SP-2001, Vector Laboratories), respectively. After incubation of primary antibodies and biotinylated secondary antibodies, tissue samples were visualized using VECTASTAIN Elite ABC HRP Kit (PK-6100; Vector Laboratories) and VECTOR NovaRed Peroxidase Substrate Kit (SK-4800, Vector Laboratories). Cell nuclei were stained using hematoxylin.

RPMI-1640 media (RPMI) were purchased from Sigma-Aldrich (St. Louis, MO); CMRL-1066 media (CMRL) were purchased from Mediatech (Holly Hill, FL); heat-inactivated fetal bovine serum (FBS) was obtained from HyClone (Logan, UT). Baicalein (98% pure) for in vitro studies was purchased from Sigma-Aldrich (St Louis, MO). Stock solutions of Baicalein at 20 mM were dissolved in sterilized dimethyl sulfoxide (DMSO) and stored at −80°C before use. Baicalein (98% pure by HPLC) for in vivo studies was purchased from Xi'An Yile Bio-Tech Company, China; ultrasensitive rat insulin enzyme-linked immunosorbent assay (ELISA) kits were obtained from Mercodia (Winston-Salem, NC); the active form of the caspase-3 antibody was from BD Biosciences (San Jose, CA); the rabbit polyclonal anti-insulin antibody was from Abcam (Cambridge, MA); the ImmPRESS Anti-rabbit Ig (peroxidase) Polymer Detection kit, Vector NovaRED peroxidase substrate kit, and Vector SG peroxidase substrate kits were from Vector laboratories (Burlingame, CA); cell viability assay kits were from Promega (Madison, WI); and the BrdU ELISA kit for the cell proliferation assay was from Roche Applied Sciences (Indianapolis, IN). All other chemicals were from Sigma-Aldrich. Glucose was dissolved in sterile water and stored at −80°C.

Formalin-fixed and paraffin-embedded human placental tissue sections were de-paraffinized in xylene and rehydrated in descending concentrations of ethanol. The tissue sections were then subjected to antigen retrieval by heating for 10 minutes at 120°C in citrate buffer (pH 6) and allowed to cool down to room temperature. The tissue sections were treated with 0.3% H₂O₂ to block endogenous peroxidases and with goat or rabbit serum to block nonspecific antibody binding sites on PLAC1 and FGFR2IIIb or FGF7, respectively. Samples were incubated with primary antibodies against PLAC1 (mouse monoclonal, mu37, Ganymed Pharmaceuticals AG), FGFR2 (rabbit polyclonal, sc-122, Santa Cruz Biotechnology), or FGF7 (polyclonal goat, AF-251-NA, R&D Systems) overnight at 4°C; the sections were washed three times with phosphate-buffered saline (PBS) and then incubated with horseradish peroxidase-conjugated secondary antibodies, goat-anti-mouse, goat-anti-rabbit, or rabbit-anti-goat (Immunologic, bv, Duiven, Netherlands), respectively. For visualization of PLAC1, FGFR2IIIb, and FGF7 localization, the Vector NovaRED peroxidase substrate kit (Vector Laboratories Inc. Peterborough, UK) was used and nuclei were counterstained with hematoxylin/eosin. Tissue sections were dehydrated and mounted (X-TRA-Kitt, Medite, Burgdorf, Germany) prior to microscopic evaluation.