Fitc conjugated donkey anti mouse igg

Rats were anesthetized and perfused with 200 ml NS and 200 ml 4% paraformaldehyde (PFA, in 0.1 M PB, pH 7.4) solution. The right eyeballs were enucleated and fixed in 4% PFA solution for 4 h, then dehydration with grade sucrose solution at 4 °C. The retinae were vertically sectioned at 10 μm thickness. Retina slices were mounted on gelatin-coated slides. The slices were blocked in 4% goat, 0.25% serum bovine serum albumin (BSA), 0.2% Triton X-100 in PBS at room temperature for 2 h, and then incubated with mouse monoclonal anti-GS (1:200 dilution; Abcam), goat polyclonal anti-GLAST (1:200 dilution; Santa cruz), rabbit monoclonal anti –Kir2.1 (1:200 dilution; Abcam), rabbit polyclonal anti-Kir4.1 (1:200 dilution; Abcam) and rabbit polyclonal anti-K_2p3.1(TASK-1)(1:200 dilution; Alomone Labs) primary antibodies at 4 °C for 48 h. Immunoreactive proteins were visualized by incubating with FITC conjugated donkey anti-mouse IgG (1:100 dilution; Jackson Immuno-Research Laboratories), FITC conjugated donkey anti-goat (1:50; Santa Cruz) and cy3-conjugated donkey anti-rabbit IgG (1:100 dilution; Biolegend). The samples were mounted with mounting medium with DAPI (Vector Laboratories) and the immunofluorescence images were visualized with a Zeiss Imager.M1 laser-scanning microscope using a 20 × objective lens.

The ascidian gonads and the COS cells were incubated overnight at 18°C (Gonads) or at 37°C (COS cells) in their respective medium containing 50 μM bromodeoxyuridine (BrdU) (sigma B5002, St Louis, MO, USA) and fixed with 1% paraformaldehyde overnight at 4°C. After 1h DNase treatment at 37°C, Gonads and COS cells were incubated with monoclonal anti-BrdU (GE Healthcare RPN 202) for 2h at 37°C. Appropriate secondary antibody was FITC-conjugated donkey-anti-mouse IgG (Jackson Laboratories). The Click-IT EdU imaging kit from Invitrogen was used as an alternative to BrdU labeling. EdU (5-ethynyl-2’-deoxyuridine) is a nucleoside analog of thymidine and is incorporated into DNA during active DNA synthesis [17 (link)]. Detection is based on a click copper-catalyzed covalent reaction between an azide and an alkyne. After dissection, gonads were opened and incubated overnight at 18°C with 50 μM EdU followed by paraformaldehyde fixation, permeabilization and DAPI staining.

Immunofluorescence staining was done according to a published protocol [2 (link)]. Primary antibodies against Aur A (sc-25425), Aur B (sc-25426), BRCA1 (sc-6954) and BRCA2 (sc-1818) were obtained from Santa Cruz Technology (California, US). DNA dye 4′,6-diamidino-2-phenylindole (DAPI) was obtained from Molecular Probes. The secondary antibodies used were the FITC-conjugated donkey anti-mouse IgG, cy3-conjugated donkey anti-rabbit IgG, Texas red-conjugated or FITC-conjugated donkey anti-goat IgG (Jackson ImmunoResearch Laboratory). All stained cells were examined and photographed with a Leica SP5 confocal fluorescence microscope.

After rehydration and antigen retrieval, the 5 μm sections were incubated with 5% donkey serum in 0.3% triton X-100 for 1 hr. Then the sections were incubated with the primary antibodies for 1.5 hrs, and the corresponding TRITC-conjugated donkey anti-rabbit IgG (1:150, Jackson) and FITC-conjugated donkey anti-mouse IgG (1:150, Jackson) for 1.5 hrs at room temperature. The following dilutions of primary antibodies were used: PRMT5 (1:200, Millipore, 07-405), DAZL (1:100, AbD Serotec, MCA2336), STRA8 (1:200, Abcam, ab49405), SCP3 (1:200, Abcam, ab15093), γH2AX (1:400, Millipore, 05-636), DMC1 (1:100, Santa Cruz, sc-22768 ), H3R2me2s (1:50, Millipore, ABE460), H4R3me2s (1:100, Abcam, ab5823). After three times wash in PBS, the nuclei was stained with DAPI. The sections were examined with confocal laser scanning microscope (Carl Zeiss Inc., Thornwood, NY).

Immunofluorescent staining of pancreata was conducted as described.^{9 (link)} Primary staining utilized the following antibodies: Eif4g (Cell Signaling Technology, Beverly, MA, USA; #8701), Os9 (Abcam, Cambridge, UK; ab109510), Tollip (Proteintech, Chicago, IL, USA; 11315-1-AP), Gsta (Thermofisher Scientific; PA5-79335), Nrf2 (Proteintech; 16396-1-AP), and Keap1 (Proteintech; 10503-2-AP), in combination with mouse monoclonal anti-insulin Abs (Sigma-Aldrich, St. Louis, MO, USA, I2018). Secondary staining utilized Alexa594-conjugated donkey anti-goat IgG, or Alexa594-conjugated donkey anti-rabbit IgG, and FITC-conjugated donkey anti-mouse IgG (all from Jackson ImmunoResearch Laboratories, West Grove PA, USA). Nuclear counterstaining utilized DAPI (Invitrogen, Waltham, MA). Images were collected using a fluorescence microscope (Keyence, Itasca, IL, USA) and analyzed with the Fiji software package for ImageJ (https://imagej.nih.gov.ij/).

Immunofluorescence staining was done according to a published protocol^{20 (link)}. Cells were treated with DMSO or chloroquine. Cells grown on cover slips were fixed in methanol for 5 min, permeabilized for 5 min with 0.3% Triton X-100 in PBS, and blocked for over 1 h with 5% bovine serum albumin and 1% goat serum. The cells were then incubated overnight at 4 °C with antibodies to ID1 (1:200), LC3B (1:200), ATF6 (1:100), or pSTAT3⁷⁰⁵ (1:100), followed by incubation with the secondary antibodies in a humid dark box at room temperature for 1 h. The secondary antibodies used were the FITC-conjugated donkey anti-mouse IgG, Cy3-conjugated donkey anti-rabbit IgG, Texas red-conjugated and FITC-conjugated donkey anti-goat IgG (Jackson ImmunoResearch Laboratory, USA). DAPI was obtained from Molecular Probes (USA). All stained cells were calculated and photographed with a Leica SP5 confocal fluorescence microscope (Leica Biosystems, GER).