Cells were fixed with 4% paraformaldehyde (4% PFA—Polysciences Inc., Hirschberg and der Bergstrasse, Germany; catalog number: 2878-55-4) in GibcoTM Dulbecco’s Phosphate-Buffered Saline (DPBS—Thermo Fisher Scientific (Brussels, Belgium), catalog number: 14190250) for 15 min and rinsed three times with DPBS. In total, 5 % normal donkey serum (NDS, Sigma, Machelen, Belgium; catalog number: D9663) in 0.1% Triton X-100 in DPBS was used for blocking at room temperature for 1 h. Primary antibodies were diluted in 2% NDS in 0.1% DPBS Triton and incubated overnight at 4 °C. The following primary antibodies were used: anti-G3BP (1/250, catalog number: ab56574), anti-Yb1 (1/500, catalog number: ab76149) and anti-importin-β1 (1/1000, catalog number: ab2811) all provided by Abcam (Cambridge, UK). The cells were subsequently washed with DPBS and incubated with appropriate secondary antibodies (1:2500; Thermo Fisher Scientific, Brussels, Belgium). Nuclei were stained with Hoechst (NucBlue Live ReadyProbesTM Reagent Hoechst 33342, Thermo Fisher Scientific, Brussels, Belgium; catalog number: R37605) and images were taken under SP8 confocal microscope (64x; Leica, Wetzlar, Germany; model: SP8 MDi8) excitation lines at 405, 488, 555 and 647 nm.
Normal donkey serum (nds)
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Italy, Australia, Belgium
Normal donkey serum is a laboratory reagent derived from the blood serum of healthy donkeys. It is commonly used in immunohistochemistry, Western blotting, and other biological assays as a blocking agent to reduce non-specific binding of antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (41 mentions)
Bovine serum albumin (bsa), by Merck Group (33 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (17 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (12 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (11 mentions)
Paraformaldehyde (pfa), by Merck Group (10 mentions)
Hoechst 33342, by Thermo Fisher Scientific (10 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (7 mentions)
Tcs sp5 confocal microscope, by Leica (7 mentions)
Triton x, by Merck Group (6 mentions)
Lsm 710 confocal microscope, by Zeiss (6 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (5 mentions)
Fluoromount g, by Southern Biotech (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (4 mentions)
Lsm 710, by Zeiss (4 mentions)
Lsm 510 confocal microscope, by Zeiss (4 mentions)
Triton x 100, by Thermo Fisher Scientific (4 mentions)
Normal goat serum (ngs), by Merck Group (4 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (4 mentions)
Porcine gelatin, by Merck Group (4 mentions)
205 protocols using normal donkey serum (nds)
1
Immunofluorescence Staining of Cellular Stress Granules
2
Immunofluorescence Analysis of Tight Junctions
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Cells were seeded into a Lab-Tek™ 8-well Chambered Coverglass (Thermo Fisher Scientific). OGD was performed on the cells once they had reached confluency. Cells were fixed and permeabilized with ice-cold methanol and acetone for 10 min and blocked using 5% normal donkey serum (Sigma-Aldrich) in PBS for 1 h. The cells were incubated with primary antibodies (anti-ZO-1 and anti-claudin-5 antibodies) diluted in 1% normal donkey serum and then further incubated with secondary antibodies (Alexa Fluor 555-conjugated anti-rabbit) in 1% BSA. Fluorescence images were acquired and analyzed with a K1-Fluo confocal laser scanning microscope (Nanoscope Systems, Daejeon, Korea). To quantify the degree of TJs proteins, fluorescence images were analyzed with the software program, ImageJ.
3
Mapping Kisspeptin and GnRH in Hypothalamus
4
Immunofluorescence Imaging of bEND.3 Cells
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The bEND.3 cells were seeded on Lab-Tek™ 8-well chambered coverslips (Thermo Fisher Scientific, Rochester, NY, USA) at a density of 9 × 103 and incubated to confluence. After MG treatment, cells were fixed and permeabilized with ice-cold methanol and acetone, respectively, blocked with 5% normal donkey serum (Sigma Aldrich) in PBS, and incubated for 1 h. The cells were then incubated with primary antibodies diluted in PBS containing 1% normal donkey serum before attaching fluorescent secondary antibodies diluted in 1% BSA for visualization. Fluorescence was recorded using a K1-Fluo confocal laser scanning microscope (Nanoscope Systems, Daejeon, South Korea), and the co-localization of the proteins was analyzed with the ImageJ plug-in JACoP (Just Another Co-localization Plugin).
5
Immunohistochemical Analysis of Tyrosine Hydroxylase in SH-SY5Y Neuroblastoma Cells
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Human SH-SY5Y neuroblastoma cells were permeabilized and fixed with 0.5% Triton X-100 and 4% paraformaldehyde. Slides were blocked with 1% normal donkey serum (Merck, Darmstadt, Germany) in phosphate buffered saline (PBS) for 60 min at room temperature. Cells were washed with 0.1% bovine serum albumin (BSA, Beyotime Institute of Biotechnology, Shanghai, China) in PBS three times with gentle shaking, then incubated with the primary antibody Tyrosine Hydroxylase (TH; 1:100, Santa Cruz, CA, USA) diluted in 0.1% BSA/PBS at 4°C overnight. Labeled donkey anti-rabbit IgG (1:1000 Invitrogen, Paisley, UK) was used as the secondary antibody and was incubated in the dark for 2 h at room temperature. The samples were subjected to chromogenic diaminobenzidine (DAB) staining. In general, one drop of A, B, C reagents was respectively added into 1 ml of distilled water. The mixture was used for cell staining. The color development was monitored under the microscope at room temperature. The reaction stopped by adding distilled water when the ideal color was developed. Hematoxylin was used as a counterstain. The samples mounted by mounting medium were visualized by an inverted microscope under 200 times and 400 times magnification.
6
Immunofluorescence Staining of AD iPSC-Derived Neurons
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The AD iPSC-derived neurons were washed with cold PBS and fixed with 4% paraformaldehyde (PFA) at room temperature for 15 min. Next, the neurons were washed three times with PBS, incubated for 1 h in blocking solution [10% normal donkey serum (Merck, Darmstadt, Germany), 2% BSA (Gemini Bio, CA, USA), 1 M glycine (Sigma Aldrich, St. Louis, Missouri, USA) in PBST (PBS + 0.1% Triton-X 100)], and incubated for 1 h with the primary antibody at room temperature. The cells were then treated with the secondary antibody diluted in blocking solution for 1 h at room temperature. After washing with PBST, the cells were incubated with Hoechst 33342 (Sigma Aldrich) for nuclear staining and mounted for imaging. Fluorescence microscopy images of the immunostained cells were obtained by an LSM 800 (Zeiss, Jena, Germany). The primary and secondary antibodies are listed in Table 2
7
Immunostaining of Lymphatic and Blood Vessels in Mouse Tissues
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Mice were euthanised, and diaphragms and ears were harvested. The tissues were subsequently washed, blocked in immunomix-2 (IM-2) containing 0.1% Triton-X (Honeywell), 1% BSA/PBS (Merck) and 0.5% normal donkey serum (Merck). Both tissues were subsequently incubated overnight at 4 °C in IM-2 with rat anti-MECA-32 antibody (5 µg/ml, Biolegend, San Diego, USA).
The following day, the tissues were incubated for 3 h with Alexa Fluor-conjugated secondary antibodies, i.e. Alexa Fluor 594 anti-rat and Alexa Fluor 488 anti-rabbit (3 µg/ml, Invitrogen, Basel, Switzerland). Diaphragms were subsequently fixed in 4% PFA for 2 h at 4 °C. Samples were washed with PBS and mounted using Mowiol (Vector Laboratories, Burlingame, USA).
Tissues were analysed on a Zeiss LSM780 inverted confocal microscope (Carl Zeiss, Oberkochen, Germany) using a 10 × 0.3 NA EC Plan-Neofluar objective and processed with the Imaris software (version 7.1.1; Bitplane, Zurich, Switzerland). In the diaphragm, images of LYVE-1+ and MECA-32+ vessels were taken in the middle part of central tendon where the distribution of both lymphatic and blood vessels is homogenous and reproducible. In the ear skin, lymphatic and blood vessels were imaged at the external rim of the ear.
The following day, the tissues were incubated for 3 h with Alexa Fluor-conjugated secondary antibodies, i.e. Alexa Fluor 594 anti-rat and Alexa Fluor 488 anti-rabbit (3 µg/ml, Invitrogen, Basel, Switzerland). Diaphragms were subsequently fixed in 4% PFA for 2 h at 4 °C. Samples were washed with PBS and mounted using Mowiol (Vector Laboratories, Burlingame, USA).
Tissues were analysed on a Zeiss LSM780 inverted confocal microscope (Carl Zeiss, Oberkochen, Germany) using a 10 × 0.3 NA EC Plan-Neofluar objective and processed with the Imaris software (version 7.1.1; Bitplane, Zurich, Switzerland). In the diaphragm, images of LYVE-1+ and MECA-32+ vessels were taken in the middle part of central tendon where the distribution of both lymphatic and blood vessels is homogenous and reproducible. In the ear skin, lymphatic and blood vessels were imaged at the external rim of the ear.
8
Immunohistochemistry of Paraffin Sections
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Paraffin embedded tissue sections (10 μm) were deparaffinized with xylene and rehydrated using a decreasing gradient of ethanol. Heat-induced antigen retrieval was performed using citrate buffer (10 mM Citric acid, 0.05% Tween-20, pH 6.0) for 20 min at 90–95°C. To reduce non-specific binding, blocking was carried out with 10% Normal donkey serum (EMD Millipore, Burlington, Massachusetts) and 0.2% Triton-X100 in PBS for 1 h at room temperature. A list of all the primary and secondary antibodies used are listed in Supplementary Table 3 . Primary antibodies were prepared in antibody diluent solution (2.5% Normal donkey serum, 0.25% sodium azide, 0.2% trition X 100, and PBS, Abcam, Cambridge, Massachusetts), and sections were covered with primary antibodies overnight at 4°C. Next, each well was washed with PBS five times and then incubated with the respective secondary antibodies for 1 h, followed by mounting with VECTASHIELD® antifade mounting medium containing DAPI (Vector Labs, Burlingame, California) on glass sides. Slides were dried overnight and then imaged using a Nikon Eclipse C1 microscope.
9
Peroxidase Immunohistochemistry for BrdU and Ki67
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Peroxidase immunohistochemistry was performed essentially as described previously.43 (link) Briefly, floating sections were incubated in hydrogen peroxide (1% in PBS) to destroy endogenous peroxidase activity; underwent antigen unmasking in 1 m HCL for 35 min at 37 °C (for BrdU only); were incubated in blocking buffer consisting of 0.2% Triton X (Sigma-Aldrich) and 5% v/v normal donkey serum (Merck-Millipore, Kilsyth, VIC, Australia) in 100 mm PBS for 1 h; were incubated in either sheep anti-BrdU (Exalpha Biologicals, Watertown, MA, USA) diluted 1/1000 or rabbit anti-Ki67 (Thermo Fisher Scientific Australia, Scoresby, VIC, Australia) diluted 1/200 in blocking buffer overnight at 20 °C; were incubated with biotinylated rabbit anti-sheep (Vector Laboratories, Burlingame, CA, USA) or biotinylated goat anti-rabbit Ki67 (Vector Laboratories) diluted at 1/500 for 2 h; incubated with Vectastain ABC solution (1:100, Vector Laboratories) for 1 h; developed with diaminobenzidine liquid chromogen kit (1:50, Dako Australia, North Sydney, NSW, Australia); were mounted on slides and then cover-slipped with DPX. Unless otherwise stated, each step occurred at room temperature and was followed by thorough washing with 100 mm PBS.
10
Quantification of Ovarian AMH Expression
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Ovaries from young (n = 4) and aged adult (n = 4) C57Bl6/J mice (separate to the main study cohort) were immersion-fixed in Bouin's fixative overnight, wax embedded, and sectioned at 5 μm thickness. The xylene-dewaxed sections were rehydrated through graded ethanol solutions and incubated in blocking solution (0.1 μg/mL in phosphate-buffered saline with 5%, normal donkey serum (Merck, Cat# D9663), 0.08% w/v Tween20 (Sigma-Aldrich, Cat# P1379) and 1% w/v bovine serum albumin) for 20 minutes. Goat anti-human AMH C-20 antibody (Santa Cruz, Cat# sc-6886, Santa Cruz, CA, USA, RRID: AB_649207) was applied at 0.1 μg/mL in phosphate-buffered saline with 0.08% w/v Tween20 (Sigma-Aldrich, Cat# P1379, Kenilworth, NY, USA) and 1% w/v bovine serum albumin, overnight incubation at 4 °C. Biotin-SP-AffiniPure Donkey Anti-Goat IgG (H + L), (Jackson ImmunoResearch Lab, Cat# 705-065-003, West Grove, PA, USA, RRID: AB_2340396) was applied at 2 µg/mL for 1 hour at room temperature followed by streptavidin-biotin-HRP complex (GE Healthcare, Cat#RPN1051, Chicago, IL, USA) diluted 1:100 for 1 hour at room temperature. Chromogen staining was generated with diaminobenzidine (DAB substrate kit, Vector Labs, Cat# SK-4100, Newark, CA, USA) with hematoxylin counterstain.
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