At the conclusion of experiments, NCD- and HFD-fed C57BL/6 mice were perfused with a 4% paraformaldehyde solution. Whole brains were removed, then stored in 4% paraformaldehyde for 3 days. After transferring brains to a 30% sucrose solution, coronal sections (25 µm thick) were prepared, washed with PBS, and then blocked by incubating for 1 hour with 3% donkey serum (Dako, Glostrup, Denmark) and 0.3% Triton X-100 in the dark. Thereafter, sections were washed with PBS and incubated overnight at 4℃ with anti-TH (1:500; Millipore, MA, USA) and anti-pJNK (1:500; Invitrogen, Carlsbad, CA, USA) antibodies, diluted in blocking solution. Sections were then washed with PBS and incubated with fluorescently labeled secondary antibody for 1 hour at room temperature. Sections from SN was obtained through -2.60 mm to -3.90 mm relative to bregma [39 (link)]. Sections from striatum was obtained through -1.08 mm to -0.84 mm relative to bregma [40 (link)]. TH and pJNK fluorescence (n=10 slides per condition, n=6 each groups) were visualized using an IX70 confocal microscope (Olympus, Tokyo, Japan).
Donkey serum
Manufactured by Agilent Technologies
Sourced in Denmark
Donkey serum is a laboratory reagent derived from the blood of donkeys. It is a complex mixture of proteins, electrolytes, and other biomolecules that can be used in various scientific applications. The core function of donkey serum is to provide a biological matrix for cell culture, immunoassays, and other in vitro experiments.
Automatically generated - may contain errors
Lab products found in correlation
Ix70 confocal microscope, by Olympus (2 mentions)
Bovine serum albumin (bsa), by Agilent Technologies (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Anti th, by Merck Group (1 mentions)
Donkey serum, by Santa Cruz Biotechnology (1 mentions)
Donkey serum, by Abcam (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
L9393, by Merck Group (1 mentions)
Pg m1, by Agilent Technologies (1 mentions)
Cd163 10d6, by Leica (1 mentions)
Biotinylated donkey anti rabbit, by Dianova (1 mentions)
Aqueous mounting solution, by Agilent Technologies (1 mentions)
Anti βiii tubulin, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by R&D Systems (1 mentions)
Anti ve cadherin, by R&D Systems (1 mentions)
Confocal software lcs, by Leica (1 mentions)
Mounting medium, by Agilent Technologies (1 mentions)
Anti th antibody, by Merck Group (1 mentions)
Envision system hrp, by Agilent Technologies (1 mentions)
Anti gfap, by Abcam (1 mentions)
11 protocols using donkey serum
1
Immunohistochemical Analysis of SN and Striatum
2
Immunofluorescence Staining of Meprin β in Mouse Kidney
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Immunofluorescence staining was performed on kidney cryosections obtained from CD1 nu/nu mice. The 3 μm thick sections were transferred onto a microscope slide, fixed in acetone for 10 min at 4°C and washed in PBS with 0.4% Triton X-100 (PBST) for 5 min. The sections were then treated with 1% donkey serum (Dako, Baar, Switzerland) in PBST, followed by blocking with 5% donkey serum in PBS for 1 h. Subsequently, the cells were incubated with polyclonal antibody directed against meprin β (sc-23491) (Santa Cruz Biotechnology, Santa Cruz, USA) diluted 1:200 in 5% donkey serum PBS for 1 h. After washing the sections with PBS they were incubated with polyclonal donkey anti-goat IgG H&L (Alexa Fluor 488) (ab150129) (Abcam, Cambridge, UK) diluted 1:600 in 5% donkey serum PBS for 1 h at room temperature. The sections were washed again and mounted with ProLong Gold antifade mountant (Life Technologies Europe B.V., Zug, Switzerland). Goat IgG, diluted 1:200 in 5% donkey serum PBS was used as primary control antibody. The fluorescent signal was visualised with a Leica SP5 Confocal Microscope (Leica Microsystems GmbH, Wetzlar, Germany) at 488 nm. The contrast of all pictures was increased to the same extent to improve the visibility of the signal.
3
Immunofluorescent Staining of Frozen Tumor Sections
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Tumor tissue samples obtained were frozen in OCT immediately and stored at −80°C. Frozen samples were sectioned at 5 mm with a cryostat, placed on slides, and air-dried. Then, the slides were fixed for 5 min with 100% acetone, pretreated with avidin/biotin blocker (DAKO) for 10 min, and blocked with 5% donkey serum (DAKO) in PBST for 30 min. Staining for CD8 (Biolegend, Clone: 53-6.7) was performed using non-labeled primary antibodies followed by Cy3 fluorophore-labeled secondary antibodies. Nuclei were then highlighted with a DAPI mounting medium.
4
Quantification of Engrafted Myoblasts
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Cryosections 8 μm thick and spaced 112 μm along the length of the muscle were collected for analysis with human-specific antibodies to measure engraftment of donor myoblasts. Frozen sections were fixed in 4% PFA for 10 min, permeabilised with 0.1% Triton X100/1% BSA (Sigma Aldrich)/PBS and blocked in 10% donkey serum (Dakocytomation) in 0.1% Triton X100/1% BSA/PBS. Cryosections were then incubated with mouse anti-human LAMIN A/C (NCL-LAM, Leica Biosystems) and mouse anti-human SPECTRIN (NCL-SPEC1, Leica Biosystems) to locate engrafted cells and myofibres, together with rabbit anti-mouse Laminin (L9393, Sigma Aldrich) to immunostain extracellular matrix and delimit myofibres. Samples were counterstained with Hoechst to identify total nuclei. The total number of donor-derived LAMIN A/C-positive nuclei within or outside of myofibres, and donor-derived SPECTRIN-positive myofibres were counted in each section along the grafted TA and expressed either as a total per mouse (all sections combined) or an average per section. Quantification was performed blinded with the experimenter unaware of whether the cells were from Sunitinib-treated or control mice.
5
Immunohistochemical Analysis of Immune Markers
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Immunohistochemistry was performed as described previously.19 (link) Briefly, after heat-induced epitope retrieval, sections (2 μm) were blocked with Donkey serum (Dako, Hamburg, Germany) and incubated with rabbit-derived antibodies specific for CD68 (PG-M1; Dako), inducible nitric oxide synthase (iNOS; polyclonal; Abcam, Cambridge, United Kingdom), TNF-α (P-T2; Abcam), CD163 (10D6; Novocastra, Berlin, Germany), or stabilin-1 (polyclonal; Sigma-Aldrich, Deisenhofen, Germany). Biotinylated donkey anti-rabbit (Dianova, Hamburg, Germany) was visualized using the REAL Detection System with alkaline phosphatase/RED (Dako). Nuclei were counterstained with hematoxylin and images acquired by light microscopy (Carl Zeiss MicroImaging, Oberkochen, Germany). Positive cells were quantified and averaged from high power fields (0.237 mm2).
6
Immunofluorescence Analysis of Endothelial Cells
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Differentiated ES cells were fixed with phosphate-buffered saline 4% paraformaldehyde (PBS-PFA) (Santa Cruz), treated for 1 h with PBS 0.1% Triton X100 (Merck Life Science) containing 5% donkey serum (Dako North America Inc) and 5% BSA (Merck Life Science) and incubated with primary antibodies for 3 h at 37 °C in a moist chamber. The following primary antibodies were used: anti-βIII-tubulin 1:1000 diluted in PBS (Merck Life Science) and anti-VE-cadherin 1: 100 diluted in PBS (R&D Systems). After rinsing three times with PBS, cells were incubated with 555 AlexaFluor donkey anti-goat and 488 AlexaFluor donkey anti-mouse (Invitrogen, Thermofisher Scientific) at 5 µg/ml for 1 h. After rinsing three times with PBS, nuclei were stained with DAPI and slides were sealed with aqueous mounting solution (Dako North America Inc). Images were captured by using a Leica TCS SPE confocal laser-scanning microscope, analyzed with Leica Confocal Software (LCS; Leica Microsystems). To quantify VE-cadherin+ endothelial cells, sequential images, covering the entire surface of a chamber slide well (0.7 cm2), were acquired with a Leica AF6000LX workstation. The area occupied by endothelial cells was measured with ImageJ software by using the Angiogenesis Analyzer for ImageJ [25 ].
7
Immunofluorescence Staining of DNA Damage
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Cells were fixed in 3.7% formaldehyde (10 min, RT), then blocked in 5% donkey serum (Dako) in PBS. Primary antibody (1:200 in PBS with 0.3% Triton X-100 and 1% BSA) was incubated overnight (4 °C, humidified chamber), cells washed twice in PBS and then incubated with secondary antibody overnight (1:1000 in PBS with 0.3% Triton X-100 and 1% BSA). Cells were washed twice in PBS before imaging. Antibodies used were α-γH2AX 9718 (CST), α-p21 Ab7960 (Abcam) and Alexafluor-568 α-rabbit IgG A11011 and Alexafluor-488 α-mouse IgG A-11011 (both Invitrogen).
8
Immunofluorescence and Immunohistochemistry of MPTP-Treated Mouse Brain
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Saline and MPTP injected Mice were perfused and fixed with 4% paraformaldehyde (PFA). The whole brain was dipped in the 4% PFA and then moved to 30% sucrose solution to dehydrate for three days. The samples were frozen and sectioned, 25 μm of each slice. For the immunofluorescence staining, after 15 min of PBS washing, sections were blocked for 1.5 h with 3% donkey serum (Dako, Glostrup, Denmark) and 0.3% triton x-100 with PBS. Then, sections were incubated with anti-TH antibody (Millipore, MA, USA), anti-GFAP (1:1000, Abcam, Cambridge, UK) diluted with blocking solution overnight at 4 °C. Sections were washed with PBS and incubated with anti-mouse Alexa 594 and anti-chicken Alexa 488-conjugated anti-IgG secondary antibodies containing solution for 1 h at room temperature. For immunohistochemistry, brain slices were incubated with anti-TH antibody for overnight at 4 °C and then incubated with a secondary antibody (Dako EnVision+ system-HRP, CA, USA) for 1 h. The slices were reacted with DAB+ substrate buffer. After mounting with mounting medium (Dako North America Inc., CA, USA), the slides were visualized using an IX70 confocal microscope (Olympus, Tokyo, Japan).
9
Immunohistochemical Analysis of GLP-1R
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Paraffin sections of kidney and pancreas tissue from normotensive WKY rats and prehypertensive SHRs were made. Sections were deparaffinated and rehydrated in double-distilled water. Sections were treated with 0.1% pronase in PBS at 37°C for 10 min and rinsed in Tris-buffered saline (TBS). Thereafter, sections were treated with 1% H 2O2 in TBS for 15 min, washed in TBS with 0.05% Tween (TBS-T), blocked with avidin for 10 min (Dako, Glostrup, Denmark), washed with TBS-T, blocked with biotin for 10 min (Dako), washed with TBS-T, and preincubated with 3.2 mg/mL poly-L-lysine, 3% BSA, 7% donkey serum, and 3% skimmed milk (Dako) for 30 min. Sections were incubated overnight with primary biotinylated mouse GLP-1R antibody, which was a generous gift from Charles Pyke (7F382A, Novo Nordisk, Måløv, Denmark). Thereafter, sections were washed three times for 10 min each in TBS-T followed by treatment with Vectastain ABComplexHRP in TBS for 30 min and washed again three times for 10 min each. Sections were developed with DAB ϩ (Dako), counterstained with hematoxylin, rinsed in water, dehydrated, and mounted.
10
Multiplex Immunohistochemistry Tissue Staining
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Sections were dewaxed and rehydrated on a Leica Multistainer ST5020. This was followed by heat induced epitope retrieval in citrate buffer (10 mM sodium citrate, pH6) in a medical pressure cooker and washing in PBS. All subsequent washes were 3 x 5 min in PBS-T (PBS with 0.05% Tween-20). After a 15 min incubation with 3% H 2 O 2 in methanol and a wash, slides were incubated with blocking buffer (PBS-T with 10% Donkey Serum, Dako) for 30 min. This and all following incubations were performed at room temperature in a humidified slide box. Slides were then incubated with the primary antibody (see table 1 in appendix) for one hour at room temperature or overnight at 4 ºC. After a wash, slides were incubated with the secondary antibody (biotin-SP-conjugated AffiniPure donkey anti-mouse, anti-rabbit or anti-goat, Jackson ImmunoResearch, all 1:500 in PBS-T) for 40 min. Following a wash, slides were incubated with Vectastain® Elite® ABC reagent (Vector Laboratories) for 40 min. This was followed by a final wash and immunoperoxidase detection using a liquid DAB + substrate chromogen system (Dako). Finally, sections were counterstained with Mayer's Haematoxylin, dehydrated and mounted in DPX on the Leica Multistainer.
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