Biotinylated anti rabbit antibody

Mice were anesthetized by isoflurane inhalation, decapitated, and their brains were rapidly removed and fixed by immersion fixation in 4% paraformaldehyde overnight at 4°C. For CRH immunostaining, mice were instead transcardially perfused with phosphate-buffered saline and 4% paraformaldehyde before immersion fixation to improve staining quality. Brains were cryoprotected in 10% and 30% sucrose in phosphate-buffered saline, rapidly frozen in isopentane on dry ice, and stored at -80°C until 40 μm coronal sections were collected using a Leica cryostat. Sections were incubated with an antibody against CRH (1:10,000, rabbit, provided by Dr. Paul Sawchenko on behalf of Dr. Wylie Vale) or c-fos (1:10,000, rabbit, Calbiochem), followed by biotinylated anti-rabbit antibody (1:200, Vector Laboratories) and streptavidin AlexaFluor 488 conjugate (1:1,000, Molecular Probes). Images were collected on a Nikon A1R confocal microscope and analyzed using ImageJ.

Prostate tissue was isolated from mice and immediately embedded in paraffin after fixation with 4% PFA at 4° C. Paraffin-embedded tissues were sectioned (5 μm thick), and de-paraffinized, and hydrated. After staining with hematoxylin and eosin (H&E) or Masson’s Trichrome (Sigma), tissues were dehydrated and mounted. The bar graph of fibrosis area was computed by dividing the area of the positive region (stained blue) by that of the fibromuscular stroma. Antigenic retrieval was performed by tissues heating hydrated tissue in 0.1 M citrate buffer (pH 6.0) in a microwave. After blocking endogenous peroxidase with H₂O₂ and inhibiting nonspecific signals with 5% goat serum, sections were incubated at 4° C overnight in a humidified chamber with a primary antibody specific for Ki67 (Abcam, Cambridge, UK). Then, sections were incubated for 1 h at room temperature with a biotinylated anti-rabbit antibody (Vector Laboratories, Burlingame, CA, USA). Next, sections were covered with ABC solution (Vector Laboratories) for 30 min. A DAB kit (Vector Laboratories) was used for chromogenic detection. Subsequent to dehydration and clearing, sections were mounted in DPX (Fluka, St. Louis, MO, USA) and observed under a light microscope (Olympus, Center Valley, PA, USA).

The first set of tissue was processed for Fos immunohistochemistry, as described previously (Worsley et al., 2007 (link)). The second and third sets were processed for pERK and pp38, respectively. In order to reduce background staining the sections were placed in 10% normal goat serum (NGS; Vector Laboratories, Peterborough, UK) made up in PBS containing 0.2% Triton X-100 (PBST) for 1 h. They were then incubated at 4 °C for 48 h with primary antibodies raised in rabbit to one of: Fos (1:20,000; Calbiochem, San Diego, CA, USA); pERK (1:600 [#9101]; Cell Signaling Technology, Danvers, MA, USA), or pp38 (1:300 [#9211]; Cell Signaling Technology) (see Table 1). In all cases, the primary antibodies were made up in PBST containing 5% NGS. The sections were then washed twice with PBS and incubated for 30 min at room temperature with a biotinylated anti-rabbit antibody raised in goat (1:300; Vector Laboratories, UK), made up in PBST containing 1.5% NGS. The labeling was visualized using the avidin–biotin complex (ABC) method with nickel-intensified diaminobenzidine (DAB) as the chromagen, as described previously (Worsley et al., 2007 (link)). Immunohistochemical controls were performed using liquid phase pre-absorption of the relevant primary antibody with the Fos peptide (Calbiochem), pERK peptide (#1150; Signaling Technology) or pp38 peptide (#1170; Cell Signaling Technology).

Retinal sections were deparaffinized and rehydrated as we previously reported40 (link). For immuno-histochemical staining, retinal sections were incubated overnight with a rabbit anti-histone GFAP antibody (1:2000 dilution, Abcam, Cambridge, UK). After extensive washing, sections were further incubated with a biotinylated anti-rabbit antibody (Vector Laboratories, Burlingame, CA). Positive staining was visualized by DAB substrate reaction (Vector laboratories) following the ABC kit protocol (Vector laboratories). High resolution pictures were taken with an Olympus BX60 microscope.

Deparaffinized pancreatic sections were incubated with TUNEL reagent (Roche Applied Science) and DAB (Life Technologies) as the color substrate. Insulin immunochemistry was then conducted by serial incubation with anti-insulin antibody (Cell Signaling Technology, 1:150), biotinylated anti-rabbit antibody (Vector Laboratories, 1:100), streptavidin-alkaline phosphatase, and then with Vector® Blue alkaline phosphatase substrate^{3 (link)} (Vector Laboratories). The percentage of TUNEL⁺ cells among total β-cells was determined in more than 30 islets per mouse (more than 180 islets per group) by manual counting under BX43 microscope (Olympus). To detect TUNEL⁺ cells among hiPSC-β-cells, TUNEL staining was followed by incubation with anti-insulin antibody and then with Alexa 594-anti-mouse IgG (Life Technologies, 1:200) which was subjected to fluorescent confocal microscopy using LSM700 microscope (Carl Zeiss). The percentage of TUNEL⁺ cells among insulin⁺ cells was determined in more than 20 islet-like clusters by manual counting. Relative β-cell mass was determined by analyzing more than 30 islets per mouse (more than 180 islets per group). After insulin immunohistochemistry using anti-insulin antibody and DAB, point counting morphometry was conducted as previously described^{3 (link)}.

At 120 min after the drug microinjection (experiment 2) or the NAC/vehicle i.p. injections (experiment 1), the animals were deeply anesthetized with isoflurane and transcardially perfused with 200 mL of PBS, followed by 300 mL of 4% formaldehyde in PB 0.1 M. The brain was removed, and 40 mm sections were obtained, as described in [24 (link)].
Selected sections were transferred to TBS and sequentially incubated (including TBS rising between incubations) in: (1) 1% hydrogen peroxide in TBS, (2) 5% goat serum in TBS-0.3% TX, (3) an anti-cFOS polyclonal antibody (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C, (4) a biotinylated anti-rabbit antibody (1:200; Vector Labs), and (5) an avidin-biotinylated peroxidase complex (1:200; ABC Elite Kit; Vector Laboratories, Inc., Burlingame, CA, USA). The reaction was visualized by incubating with diaminobenzidine (SigmaFAST, Sigma, St. Louis, MO, USA). Finally, the sections were mounted on slides, dehydrated in alcohols, cleared, and coverslipped for microscopical examination.