Methylbutane

For histology, rat eyes were embedded in Tissue Tec OCT (Paesel and Lorey) and snap frozen in methyl butane (Merck). Air-dried cryosections (8 µm) were post-fixed in ice-cold acetone for 10 min, stained with hematoxilin (Merck), and graded to obtain a pathology score as described (36 (link)). For immunofluorescence staining, acetone-fixed sections were pre-incubated with PBS containing 3% normal rabbit serum and 3% donkey serum for 15 min at room temperature (RT), washed once with PBS, and then incubated with rabbit anti-rat FoxP3 antibody (Novus Biologicals, Abingdon, UK; diluted 1:500 in PBS), mouse anti-rat TCR-ab-FITC clone R73 (eBioscience, diluted 1:40), or mouse anti-rat TCR-gd-FITC clone V65 (Aviva Systems, diluted 1:6) for 1 h at RT in the dark. Control stainings were performed with secondary antibody only. After PBS washes, Cy3-conjugated donkey anti-rabbit IgG(H + L) (Jackson Laboratories) was added (1:100 in PBS) and incubated for 1 h at RT in the dark. The slides were washed, mounted with Entellan (Merck), imaged with a Zeiss Axioskop 2plus (Carl Zeiss), and photographs taken with a Sony CyberShot DSC-S70 3.3 mp digital camera.

The perfusion-fixed brains were cryoprotected in 20% sucrose (Merck) solution and then frozen in dry ice-cold methyl-butane (Merck). Free-floating 50 μm thick serial coronal sections of the hypothalamus were cut by a frigomobil (Frigomobil, Reichter-Jung, Vienna, Austria). Immunohistochemistry was performed using standard protocols. For cFos immunostaining we used a rabbit anti-cFos (1:20,000, Cat. # ABE 457, Merck) primary antibody. The cFos signal was developed by using the standard ABC method and nickel-diaminobenzidine as chromogen. For BrDU and nesfatin-1 double fluorescence immunostaining, the sections were treated with 2N HCl at 37°C for 30 min to perform antigen retrieval. The following primary antibodies were used: mouse anti-BrDU (1:1,000, Cat. # B8434, Merck) and rabbit anti-nesfatin-1 (1:6,000, Cat. # H-003-22, Phoenix, recognizes also NUCB2 protein). The BrDU and nesfatin-1 immunostainings were visualized sequentially by using TSA Kits containing Alexa Fluor 488 and 568 fluorophores, respectively (Cat. # T20912, T20924, Thermo Fisher Scientific). Microwave treatment in 0.1 M citric acid at pH 6 was used to eliminate the activity of the secondary antibody conjugated peroxidase enzyme between the two detection steps (Toth and Mezey, 2007 (link)).

For immunohistochemistry experiments, different hearts were dissected into left and right ventricles and fixed by immersion in 1% paraformaldehyde (w/v) in PBS at 4°C for 1 h. Fixed tissue was then washed and cryoprotected by immersing it through a series of sucrose solutions (series of 10, 20 and 30% w/v). Excess sucrose solution was removed before a thin layer of O.C.T. compound (Tissue-Tek) was applied to coat the tissue. The tissue was snap-frozen for 2 min by immersion in methylbutane (Sigma) within a container of liquid nitrogen. Frozen tissue blocks were cryosectioned with a Feather Blade at −20°C. Ten microlitre-thick sections were obtained and attached to coverslips until immunofluorescent labelling.
Cardiac tissue sections were treated with Image-iT FX signal enhancer (Thermo Fisher Scientific) for 1 h at room temperature (20–22°C) prior to incubation with primary antibodies (RyR: MA3-916 (Thermo Fisher Scientific); sodium–calcium exchanger: R3F1 (Swant); caveolin-3: 610420 (BD Transduction)), overnight at 4°C, and diluted 1 : 200 in incubation solution. After washing in PBS, sections were then incubated with secondary antibodies (as above) for 2 h at room temperature, in incubation solution.