Alexa 647 conjugated streptavidin

Sections were incubated in citrate buffer (10 mM citric acid, 0.05% Tween 20 [pH 6.0]) for 15 min at 90°C, allowed to cool down, washed with PBSTx (PBS containing 0.1% Triton X-100), blocked with 5% BSA in PBSTx for 30 min at room temperature, and incubated with the primary antibody over night at 4°C (see list of antibodies below). After PBSTx washes, incubation with Alexa647- and/or Alexa555-conjugated secondary antibodies (Molecular Probes; 1/500 in PBSTx with DAPI) was performed for 1 h at room temperature. After PBSTx washes, the slides were mounted with Fluoro-Gel (Electron Microscopy Sciences). For TUNEL staining, endogenous biotin was blocked after antigen retrieval using the Avidin/Biotin blocking kit (Vector #SP-2001), and TdT enzyme and Biotin-16-dUTP (Sigma #3333566001 and #11093070910) were subsequently used following manufacturer instructions. Biotin-tagged DNA nicks were revealed with Alexa488- or Alexa647-conjugated streptavidin (Molecular Probes, 1/1000) during the incubation with the secondary antibody.
Antibodies (host species, vendor, catalogue number, dilution) included tdT (rabbit polyclonal, Rockland #600-401-379, 1/500), p21 (rabbit polyclonal, Santa Cruz Biotechnology #sc-471, 1/300), p19^Arf (rat monoclonal, clone 12-A1-1, Novus Biologicals #NB200-169, 1/100), and p16-INK4A (rabbit polyclonal, Proteintech #10883-1-AP, 1/300).

After recording the physiological responses of EB ring neurons, biocytin hydrazide was iontophoresed into the cell with a constant hyperpolarizing current of 0.9–1.2 nA passed for at least 5 min. The brain was then fixed in 4% paraformaldehyde in PBS overnight at 4°C. After washing for 1 hr in several changes of PBST (0.3% Triton X-100 in PBS) at room temperature, brains were incubated with Alexa-647 conjugated Streptavidin (1:300, Molecular Probes, ThermoFisher Sci.) in PBST overnight at 4°C. After extensive washing (15 min, 3 times in PBST), brains were processed following standard fluorescent immunostaining protocol.

All recorded cells were filled with biocytin and processed for immunhistochemistry. After the recordings, slices were fixed in 0.1 M phosphate buffer (PBS) containing 4% paraformaldehyde at 4 °C overnight. After fixation, slices were gelatin embedded and re-sectioned at 50 μm thickness. Slices were then rinsed in PBS, followed by antigen retrieval with 0.01 M sodium citrate for 10 min, and pre-incubation with 10% horse serum in 0.1 M TBS for 2 h. Immunopositivity for KV10.2 was tested with a primary antibody raised in rabbit (1:1000, Alomone Labs, Cat# APC-053, RRID: AB_2039935, rabbit, polyclonal). Further immunolabeling was used against ROR beta (1:1000, RORbeta (K-16); Santa Cruz, Cat#sc-21354, RRID: AB_2180298; goat, polyclonal). The following secondary antibodies were used to visualise the immunoreactions: Alexa 488-conjugated donkey anti-rabbit (1:200, Thermo Fisher Scientific, Cat# R-37118), Alexa 647-conjugated donkey anti-goat IgG1 (1:200, Thermo Fisher Scientific, Cat# A-21447). Biocytin labelling was visualised with Alexa 647- conjugated streptavidin (1:200, Thermo Fisher Scientific, Cat# S11225). High magnification fluorescent images were acquired with a fully automated Zeiss LSM 710 confocal microscope, using ×20 and ×63 objectives.

Diabetes was induced in Rip2-CRE/floxedSTOP-Kir6.2-V59M double heterozygous mice at 12–13 weeks of age. Pancreata were collected 2 weeks after induction, fixed in 4% paraformaldehyde and embedded in paraffin. Paraffin sections were rehydrated and subjected to heat-induced epitope retrieval (10 mM citric acid pH6/NaOH, 90–92 °C, 5 min), then permeabilised (PBS 0.5% triton X-100). Prior to staining, sections were incubated in blocking buffer (10% goat serum, 1% BSA, 0.1% Triton X-100 in PBS). Glycogen was detected using mouse anti-glycogen (ref. ^{58 (link)}, made in house; dilution 1:200), biotin-conjugated goat anti-mouse secondary antibody (BA-9200, Vector Labs, 1:200) and Alexa647-conjugated Streptavidin (S32357, Thermo Fisher Scientific, 1:500). Insulin was detected using guinea pig anti-insulin (A0564, Dako 1:500) and Alexa488-conjugated goat anti-guinea pig secondary antibody (A11073, Thermo Fisher Scientific, 1:500). All staining antibodies and conjugates were diluted in blocking buffer. Nuclear counterstain was performed with SYTOX blue (S11348, Thermo Fisher Scientific, 1:2000). Glycogen and Insulin were quantified by measuring the average fluorescence density (F/A) within the insulin-positive area of the islet, then subtracting background F/A in a same-size adjacent section of tissue. Data were normalised to the average F/A measured for control islets.