Flash frozen liver tissue samples were embedded in Tissue Freezing Medium® (Leica, Wetzlar, Germany). Then, 5 µm liver sections were fixed in methanol for 15 min at −20 °C and blocked in blocking buffer based on 5% w/v donkey serum (Jackson Immunoresearch Inc., West Grove, PA, USA) during 1 h. Subsequently, liver sections were incubated with primary antibodies directed against E-cadherin, β-catenin and γ-catenin (Abcam, Cambridge, UK; Cell Signaling Technolgy, Danvers, MA, USA) in blocking buffer based on 5% w/v donkey serum (Jackson Immunoresearch Inc., West Grove, PA, USA) overnight at 4 °C. Primary antibodies used for immunohistochemistry analysis are presented in Table 2 . After extensive rinsing with phosphate-buffered saline supplemented with 0.5% Tween-20, samples were incubated with polyclonal goat anti-rabbit Alexa Fluor® 488-conjugated secondary antibody (Jackson ImmunoResearch Inc., West Grove, PA, USA) in blocking buffer based on 5% w/v donkey serum (Jackson ImmunoResearch Inc., West Grove, PA, USA). Thereafter, Vectashield with 4′,6-diamidino-2-phenylindol (Vector laboratories, Burlingame, CA, USA) was used to stain nuclei and as mounting medium. Detection was performed using a fluorescence microscope Nikon Eclipse Ti (Nikon, Tokyo, Japan).
Donkey serum
Manufactured by Jackson ImmunoResearch
Sourced in United States, Panama, United Kingdom, Cameroon, Germany
Donkey serum is a biological product derived from the blood of donkeys. It is a complex mixture of proteins, antibodies, and other biomolecules. Donkey serum can be used as a component in various laboratory procedures and assays, such as cell culture media, immunoassays, and other biochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (52 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (38 mentions)
Bovine serum albumin (bsa), by Merck Group (23 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (23 mentions)
Hoechst 33342, by Thermo Fisher Scientific (21 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (17 mentions)
Paraformaldehyde (pfa), by Merck Group (15 mentions)
Lsm 710, by Zeiss (15 mentions)
Lsm 880, by Zeiss (14 mentions)
Lsm 780 confocal microscope, by Zeiss (12 mentions)
Donkey serum, by Merck Group (10 mentions)
Triton x 100, by Thermo Fisher Scientific (9 mentions)
Lsm 880 confocal microscope, by Zeiss (8 mentions)
Donkey serum, by Thermo Fisher Scientific (8 mentions)
Lsm 700 confocal microscope, by Zeiss (7 mentions)
Hoechst 33342, by Merck Group (6 mentions)
Lsm 700, by Zeiss (6 mentions)
Hoechst, by Thermo Fisher Scientific (6 mentions)
Alexa fluor 488 donkey anti rabbit, by Thermo Fisher Scientific (6 mentions)
Axio observer z1, by Zeiss (6 mentions)
307 protocols using donkey serum
1
Immunohistochemical Analysis of Liver Tissue
2
BRN3A Whole Mount Immunostaining
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Whole mount immunostaining for BRN3A was performed as we recently described [18 (link)]. Briefly, fixed eyecups were incubated in PBS buffer containing 0.5% Triton-X100 and 2% donkey serum (Jackson ImmunoResearch Lab, MS) for 1.5 h at room temperature. They were then transferred into the same buffer containing mouse anti-BRN3A (Millipore, Cat.#MAB1585, 1:50) and incubated overnight at 4 °C. The eyecups were thoroughly rinsed in PBS buffer with 0.5% Triton-X100; they were then fixed for an additional 10 min in 4% paraformaldehyde and rinsed again. The eyecups were whole-mounted onto Fisher Plus slides and incubated in 2% Triton-X100 and 2% donkey serum with a secondary antibody (Alexa-594-conjugated donkey-anti-mouse, 2 μg/ml, Jackson ImmunoResearch Lab, MS) for 2 h at room temperature. The whole mounts were rinsed in PBS buffer and stained with 300 ng/ml DAPI for 5 min at room temperature. After a final wash with PBS buffer, the slides were coverslipped with Immu-Mount (ThermoFisher) and used for fluorescence microscopy. Images were acquired with a Nikon fluorescence microscope using a 20× objective lens and analyzed by Nikon Elements software.
3
Immunohistochemical Staining of Oxytocin and Vasopressin Neurons
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Brain sections (40 μm) including the paraventricular nuclei (PVN) region (bregma − 0.6 to − 2.0 mm Anterior–Posterior (A–P) in rats, bregma − 0.6 to 1.2 mm A–P in mice) were collected and alternate sections were designated for OXT or AVP staining. A total of 9–10 (rats) and 4–6 (mice) sections spanning the entire PVN were stained for either OXT or AVP. Sections were washed (3 × 10 min each in 1×PBS, 0.05% Triton X-100), blocked and permeabilized for 1 h in 5% donkey serum (Jackson ImmunoResearch, West Grove, PA, USA), 0.5% Triton X-100 in 1×PBS and stained with anti-oxytocin PS38 mouse monoclonal antibody or anti-vasopressin PS41 mouse monoclonal antibody (a gift from Dr Harold Gainer, NIH, Bethesda, USA)98 (link) (1:500 in 5% donkey serum and 0.5% Triton X-100 in PBS) then left overnight at 4 °C. The following day, sections were washed and incubated in Alexa Fluor 594 Donkey anti-Mouse IgG or Alexa Fluor 488 Donkey anti-Mouse IgG (for OXT-Cre, AAV1-SYN-CRE, and AAV1/2-SYN-tdTomato experiments) (1:1000 in 0.5% Triton X-100 in PBS; cat. no. A-21203; ThermoFisher Scientific, MA, USA) for 1 h at room temperature. Sections were then washed and mounted with VECTASHIELD Antifade Mounting Medium with DAPI (cat. no. H-1200, Vector Labs, Burlingame, CA, USA).
4
Immunohistochemical analysis of neuronal markers
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Sections were blocked in 1x PBS - 0.3% Triton containing 3% donkey serum (Jackson ImmunoResearch, West Grove, USA), for 1 h at room temperature followed by incubation in primary antibody solution: chicken anti-TH (1:1000; Millipore, USA) or rabbit anti c-Fos (1:500; Santa Cruz Biotechnology, Dallas, TX, USA) in 1x PBS - 0.1% Triton containing 3% donkey serum for 48 h at 4 °C. Sections were then washed 4 times (10 min each) with 1x PBS and immediately transferred to secondary antibody solution: AlexaFluor 647-conjugated donkey anti-chicken (1:1000; Jackson ImmunoResearch, West Grove PA, USA) or Cy3 donkey anti-rabbit (1:500, Jackson ImmunoResearch, West Grove, PA, USA) and containing a DNA-specific fluorescent probe (DAPI; 1:50,000) in 1x PBS containing 3% donkey serum for 2 h at room temperature. Sections not processed for immunohistochemistry were incubated in 1x PBS - 0.3% Triton containing 3% normal donkey serum (Jackson ImmunoResearch, West Grove, USA) and DAPI (1:50,000) for 1 hr. Sections were washed 4 times (10 min each) in 1x PBS and mounted onto glass slides. Slices were allowed to dry and were coverslipped using polyvinyl alcohol (PVA) mounting medium with DABCO (Sigma, MO, USA). Stereotaxic coordinates were determined using brain atlases for rat61 and mouse62 .
5
Lysosomal Localization of mTOR in HeLa Cells
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For the MTOR lysosomal localization experiment, HeLa cells were cultured on coverglass bottom dishes (NEST, 801001) for 24 h. Cell plates were washed twice with PBS, starved in amino acid-free DMEM supplemented with insulin for 60 min, and then left untreated or stimulated with amino acids for 20 min. After that, cells were aspirated medium, washed with PBS twice, and fixed for 10 min with 4% paraformaldehyde in PBS at RT. Cell dishes were then rinsed 3 times with PBS and permeabilized with 0.1% Triton X-100 for 20 min at RT. Cells were subsequently incubated with blocking buffer (0.01% Triton X-100 plus 10% donkey serum [Jackson ImmunoResearch, 017-000-121] and 2% BSA in PBS) for 1 h at RT and then incubated with primary antibodies (LAMP2 and MTOR) in 5% donkey serum at 4°C overnight. Cells were then rinsed five times with PBS and incubated with secondary antibodies in 5% donkey serum for 2 h at RT. After washing, cells were incubated with DAPI (Roche, 28718-90-3) for 10 min. Images were detected on Nikon A1 confocal microscopes using a 60×oil immersion objective. Colocalization analysis was performed using Fiji 1.0 software.
6
Immunofluorescence Microscopy Protocol
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Cells were incubated with 5% donkey serum (Jackson ImmunoResearch Laboratories) in PBST (PBS + 0.1% Triton-X) for 30 min to avoid nonspecific antibody binding and permeabilize the cells. Primary antibodies, diluted in 5% donkey serum/PBST, were then incubated at 4 °C overnight with shaking. After primary antibody incubation, cells were washed three times with PBST and donkey secondary antibodies conjugated with Alexa Fluor 488 (i.e., Anti-Goat, Anti-Rabbit, Anti-Chicken, Anti-Mouse) or TRITC (i.e., Anti-Goat, Anti-Rabbit, Anti-Chicken, Anti-Mouse, Anti-Rat) (Jackson ImmunoResearch Laboratories) diluted at 1:400 in 5% donkey serum/PBST were added to the cells for 30 min at RT. Then, cells were thrice washed with PBST and nuclei were stained with DAPI (Roche Diagnostics). Antibody sources, catalog numbers, and dilutions are listed in Supplementary Table 2 . For imaging, we used Leica DMI6000 or confocal Leica TCS SPE. Images were initially processed by LAS X software and then further analyzed and quantified using ImageJ software (NIH, W Rasband, http://rsb.info.nih.gov/ij ) using cell counter plug-in. Typically, at least five randomly selected images were counted per condition and per replicate.
7
Quantifying Tumor Angiogenesis via CD31
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Tumor cryosections (5 μm thick) were analyzed for CD31 (specific marker for endothelial cells) as we described in the study by Filleur et al.10 (link) Cryosections were fixed for 10 min in acetone, 1 : 1 acetone/chloroform and acetone; and blocked with 2% donkey serum (Jackson ImmunoResearch, West Grove, PA, USA). The sections were then incubated with CD31 Ab (1/125, BD Pharmingen, San Jose, CA, USA) in 2% donkey serum, followed by donkey anti-rat rhodamine X (1/200, Jackson ImmunoResearch). Fluorescent images were obtained using a Zeiss LSM 510 MetaLaser Scanning confocal microscope and quantified with MetaView software. Microvessel density was defined as the average area corresponding to the CD31-positive structures counted in 10 high-powered fields per experiment condition.
8
Isolation and Immunofluorescence of Murine Dermal Explants
9
Lysosomal Localization of mTOR in HeLa Cells
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For the MTOR lysosomal localization experiment, HeLa cells were cultured on coverglass bottom dishes (NEST, 801001) for 24 h. Cell plates were washed twice with PBS, starved in amino acid-free DMEM supplemented with insulin for 60 min, and then left untreated or stimulated with amino acids for 20 min. After that, cells were aspirated medium, washed with PBS twice, and fixed for 10 min with 4% paraformaldehyde in PBS at RT. Cell dishes were then rinsed 3 times with PBS and permeabilized with 0.1% Triton X-100 for 20 min at RT. Cells were subsequently incubated with blocking buffer (0.01% Triton X-100 plus 10% donkey serum [Jackson ImmunoResearch, 017-000-121] and 2% BSA in PBS) for 1 h at RT and then incubated with primary antibodies (LAMP2 and MTOR) in 5% donkey serum at 4°C overnight. Cells were then rinsed five times with PBS and incubated with secondary antibodies in 5% donkey serum for 2 h at RT. After washing, cells were incubated with DAPI (Roche, 28718-90-3) for 10 min. Images were detected on Nikon A1 confocal microscopes using a 60×oil immersion objective. Colocalization analysis was performed using Fiji 1.0 software.
10
Dermal Explant Immunostaining Protocol
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Dermal explants were isolated from 12-week-old pregnant female ICR mouse ears (mice obtained from Harlan Laboratories) and were prepared by separating the dorsal and ventral surfaces as described by Lammermann et al.57 (link) Back skin was isolated from E17.5 ICR mouse embryos (with morning of plug discovery counted as E0.5; also from Harlan). All mice were housed, bred and killed according to an approved NIDCR animal study protocol. Sample were not fixed or permeabilized. Nonspecific protein-interaction sites were blocked with 20% donkey serum (Jackson Immuno-Research Laboratories) together with mouse on mouse reagent (M.O.M.; Vector Laboratories) in PHEM buffer for 1 h. Primary and secondary antibody Alexa-fluor 488 donkey anti-rabbit, Jackson Labs) were mixed with 10% donkey serum in PHEM buffer and incubated for 1 h with 5 washes of PHEM buffer between labelling steps. Imaging was carried out using a LSM 510 NLO META confocal microscope (Carl Zeiss Inc.) equipped with a Apochromat × 63 1.2 NA water objective and a 488-nm Argon laser was used to provide excitation.
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