Biopsies of dorsal skin were isolated and fixed overnight in 4% paraformaldehyde, washed with PBS, dehydrated, paraffin embedded, and sectioned at 5 μm. For antigen retrieval, the sections were incubated with 10 mM sodium citrate buffer, within a closed plastic container placed in a boiling waterbath, for 20 min. Sections were permeabilised with 3 × 5 min 0.1% Triton in PBS, blocked for 1 hr in 5% skim milk in PBS, and incubated overnight in 5% milk with primary antibodies. The following antibodies were used: goat anti-Dct at dilution of 1/1000 (Santa Cruz Biotechnology, sc-10451) and rabbit anti-Sox10, at 1/2000 (Abcam, ab155279). Sections were washed 3 × 5 min 0.1% Triton in PBS, and incubated with secondary antibodies, Alexa 488 donkey-anti-goat, and Alexa 555 donkey-anti-rabbit (Invitrogen) for 2 hr. Sections were subsequently incubated with 1/2000 Hoechst nuclear stain for 10 min, washed 3 × 5 min in PBS, dried and mounted with Vectashild.
Alexa 488 donkey anti goat
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Alexa Fluor 488 donkey anti-goat is a fluorescently-labeled secondary antibody used for detection and visualization in immunoassays and other fluorescence-based applications. It is designed to bind to primary antibodies raised in goat, allowing for specific labeling and detection of target antigens.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 555 donkey anti rabbit, by Thermo Fisher Scientific (5 mentions)
Donkey anti mouse alexa 488, by Thermo Fisher Scientific (5 mentions)
Donkey anti rabbit alexa 594, by Thermo Fisher Scientific (5 mentions)
Donkey anti mouse alexa 594, by Thermo Fisher Scientific (3 mentions)
Lsm 700, by Zeiss (2 mentions)
Goat anti rabbit alexa 488, by Thermo Fisher Scientific (2 mentions)
Dako envision kit, by Agilent Technologies (2 mentions)
Cy3 conjugated goat anti mouse igg, by Jackson ImmunoResearch (2 mentions)
Alexa 594 donkey anti rat, by Thermo Fisher Scientific (2 mentions)
Donkey anti rabbit alexa 647, by Thermo Fisher Scientific (2 mentions)
Slowfade gold mounting medium with dapi, by Thermo Fisher Scientific (2 mentions)
Goat anti gfp, by Rockland Immunochemicals (2 mentions)
Nb600 493, by Novus Biologicals (2 mentions)
Donkey anti rabbit alexa 488, by Thermo Fisher Scientific (2 mentions)
Goat anti mouse alexa 488, by Thermo Fisher Scientific (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Ab155279, by Abcam (1 mentions)
Anti collagen 4 antibody, by Southern Biotech (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Mab360, by Merck Group (1 mentions)
34 protocols using alexa 488 donkey anti goat
1
Immunohistochemical Analysis of Skin Biopsies
2
Immunostaining of Cells on Coverslips
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Cells were seeded on 10-mm glass coverslips, fixed with 4% paraformaldehyde, and blocked/permeabilized with 0.1% Triton X-100/3% bovine serum albumin. Cells were incubated with the indicated primary antibodies. The secondary antibodies used were Alexa488–donkey anti-mouse and Alexa488–donkey anti-goat (Invitrogen). The cells were washed extensively with PBS between antibody incubations. The coverslips were mounted with immunofluorescence mounting medium. Images were recorded on a CLSM (Zeiss LSM 700) confocal microscope.
3
Quantifying Intraepidermal Nerve Fibers
4
Fluorescent Immunohistochemistry for Protein Localization
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Fluorescent immunohistochemistry was performed on cryostat-sectioned brains. Sections were washed in PBS followed by primary antibody incubation overnight. All antibodies were diluted in supermix. Anti-MFSD1 (1:50) and anti-MFSD3 (1:50) were used to label the proteins. They were co-stained with the neuronal nuclear marker anti-NeuN (1:400 mouse, cat. no: MAB377, Millipore) (Mullen et al. 1992 ), the astrocyte marker anti-GFAP (1:400 mouse, cat. no: MAB360, Millipore) (Reeves et al. 1989 (link)) and the dendritic marker anti-MAP2 (1:400 mouse, cat. no: M4403, Sigma-Aldrich) (Izant and McIntosh 1980 (link)). After additional PBS washes, slides were incubated with secondary antibody for 2 h at RT. Secondary antibodies used are as follows: alexa 488 goat-anti-rabbit, alexa 488 donkey-anti-goat and alexa 594 donkey-anti-mouse (Invitrogen) diluted 1:400. PBS washes followed by 10 min DAPI staining (1:3000 in PBS, Sigma-Aldrich) and additional washes before mounted in Mowiol anti-fade media (25 g Mowiol 4–88 in 100-ml 1× PBS, pH 8.0, 50 ml glycerol, 3 ml of 1 % thimerosal and 100 μg/ml n-propyl gallate in (Sigma-Aldrich)). Images were taken using an Olympus microscope BX53 with an Olympus DP73 camera. The micrographs were acquired by cellSens Dimension software.
5
Neuronal Excitability and Morphology Analysis
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The membrane excitability of the recorded neurons was measured in current-clamp mode by determining the number of action potentials elicited by intracellular injection of 0, 10, 20, 30, 40, 50, and 60 pA depolarizing currents for 400 ms. The spike number was determined to estimate the influence of rapamycin on the recorded neurons.
In all cases, biocytin (0.5%) was introduced into the intracellular solution to identify the morphological properties of the recorded neurons. After recording, the brain slices were immediately fixed in 4% paraformaldehyde in 0.1 M PB for 4 h at room temperature. Then, sections were rinsed with 3% hydrogen peroxide in 0.01 M PBS for 30 min. After thorough washing with PBS, the tissue was incubated with a goat anti-5-HT (1 : 500, Immunostar) antibody in PBS-NDS (pH 7.4) for 24 h, followed by incubation with Alexa 594 avidin D (1 : 1000, Invitrogen) and Alexa 488 donkey anti-goat (1 : 500, Invitrogen) antibodies in PBS for 6 h at room temperature. The sections were then observed, and images were captured with a confocal microscope (Olympus).
In all cases, biocytin (0.5%) was introduced into the intracellular solution to identify the morphological properties of the recorded neurons. After recording, the brain slices were immediately fixed in 4% paraformaldehyde in 0.1 M PB for 4 h at room temperature. Then, sections were rinsed with 3% hydrogen peroxide in 0.01 M PBS for 30 min. After thorough washing with PBS, the tissue was incubated with a goat anti-5-HT (1 : 500, Immunostar) antibody in PBS-NDS (pH 7.4) for 24 h, followed by incubation with Alexa 594 avidin D (1 : 1000, Invitrogen) and Alexa 488 donkey anti-goat (1 : 500, Invitrogen) antibodies in PBS for 6 h at room temperature. The sections were then observed, and images were captured with a confocal microscope (Olympus).
6
Immunohistochemical Analysis of Skin Biopsies
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Biopsies of dorsal skin were isolated, cut into small pieces, fixed overnight in 4% paraformaldehyde, washed with PBS, dehydrated, paraffin imbedded and sectioned at 5 μm. For antigen retrieval, the sections were incubated with 10mM sodium citrate buffer, within a closed plastic container placed in a boiling waterbath, for 20 min. Sections were permeabilised with 3x5 min 0.1% Triton in PBS, blocked for 1h in 5% skin milk in PBS, and incubated overnight in 5% skin milk with primary antibodies. The following antibodies were used: goat anti-Dct at dilution of 1/1000 (Santa Cruz Biotechnology, sc-10451), rabbit anti-Ki67, at 1/500 (Novocastra Laboratories, NCL-Ki67p), rabbit anti-Sox10, at 1/1000 (Abcam, ab155279), H3K4me3 (04–745 Millipore), H3K9ac (07–352 Millipore), H3K27me3 (CS200603 Millipore), H3K27ac (39133 Active motif) SMARCA5 (Abcam ab72499). Sections were washed 3x5 min 0.1% Triton in PBS, and incubated with secondary antibodies, Alexa 488 donkey-anti-goat, and Alexa 555 donkey-anti-rabbit (Invitrogen) for 1 h. Sections were subsequently incubated with 1/2000 Hoechst nuclear stain for 10 min. Sections were washed 3x5 min in PBS, dried, mounted with Vectashild, and coverslip immobilized with nail polish.
7
Immunofluorescence Analysis of MEIS1, SOX2, and KI67
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Samples were deparaffinised, rehydrated and permeabilised as previously described (Sanz-Navarro et al., 2018 (link)). Anti-MEIS1 primary antibody was used at a dilution of 1:1000 (rabbit, Abcam, ab19867), anti-SOX2 at 1:200 (goat, Santa Cruz, SC-17320) and anti-KI67 at 1:200 (rabbit, Abcam, ab16667). Secondary antibodies Alexa488 donkey anti-goat (Invitrogen) and Alexa568 goat anti-rabbit (Invitrogen) were used at 1:400. Samples were counterstained by incubating with Hoechst (1:1000, Invitrogen). Results were imaged by means of fluorescent microscopy (BX61, Olympus) and images were processed using Adobe Photoshop.
8
Optogenetic Stimulation and c-Fos Imaging
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Rats were placed into a clean rat cage, and a 200-µm diameter fibre-optic patch cord (Doric), coupled to a 473-nm (Omicron) laser, was connected to the implanted optical fibre. Immediately before this, the power output from the patch cord was adjusted to 20 mW. Stimulated animals received 10 Hz, 10-ms pulse width and 473-nm stimulation for 10 min before being returned to their homecage. Unstimulated animals received no stimulation for 10 min before being returned to their homecage. Rats were euthanized by transcardial perfusion with 150 ml PBS, followed by 100 ml 4% PFA 90 min after optogenetic stimulation. Brains were extracted and 100-μm sections were cut on a vibratome. The slices were labelled with goat anti-GFP (abcam) and rabbit anti-FOS (abcam) primary antibodies, Alexa 488 donkey anti-goat (Invitrogen) and Alexa 594 donkey anti-rabbit (Invitrogen) secondary antibodies, and DAPI. Images were acquired using a confocal microscope (Zeiss) with a ×20 objective and overlaid with images from the Paxinos, George and Watson rat brain atlas for blinded manual counting of FOS-positive cells in specified brain regions.
9
Peptide Competition Assay for DCX Antibodies
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To determine the specificity of the goat anti-DCX antibody (Santa Cruz, sc-8066) and the guinea-pig anti-DCX antibody (Chemicon, AB5910) a peptide competition assay was performed. The DCX peptide was no longer available at Chemicon. As such, both antibodies were submitted to a competition assay with the DCX peptide provided by Santa Cruz Biotechnology (sc-8066). A 1:5 solution of anti-DCX antibody and anti-DCX peptide (sc-8066P, Santa Cruz) was incubated at room temperature for 1 h. The preparation was then diluted in 0.3% Triton and each section was covered with 50 μl and incubated at room temperature for 3 h. Following incubation, slides were washed successively three times for 5 min in PBS (10 mM). Each section was then incubated for 30 min at 37°C with 50 μl of the secondary antibody Alexa488 donkey anti-Goat (1:1,000, Invitrogen) or Alexa488 anti-guinea pig (1:500, Jackson Immuno Research) diluted in 0.3% Triton in 10X PBS. After incubation, slides were washed successively three times for 5 min in 10X PBS. Sections were imbedded in custom-made anti-fade solution (p-Phenylenediamine and glycerol in PBS solution) and cover-slipped with micro cover glasses (VWR Scientific).
10
Immunofluorescence Staining of Stem Cells
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On day 5 post seeding, assay plates were washed 1× with PBS and fixed for 5 min at RT with 4% PFA, rinsed 1× in PBS and 2× in PBST (PBS + 0.1% Tween20) and permeabilized for 10 min in PBS containing 0.1 M Glycin/0.1% Triton X-100. After permeabilization, cells were rehydrated for 10 min in PBST containing 0.1 M Glycin without Triton X-100 and blocked for 30 min with PBST+ 10% FCS, 0.1% BSA, 3% donkey serum. Primary antibodies were diluted in blocking solution (anti-FoxA2 goat polyclonal AB (Santa Cruz, sc6554) and anti-Oct-3/4 mouse monoclonal AB (Santa Cruz, sc5279), (both 1:800 diluted). Plates were incubated with 20 μl of the antibody dilution for 1 h at 37 °C, rinsed with 1× with PBST and washed 2× for 7 min in PBST. Secondary antibodies (Alexa555 anti-mouse IgG and Alexa488 donkey anti-goat, Invitrogen) were diluted in blocking solution (1:800). Secondary antibody solution was added to each well and incubated for 1 h. Secondary antibody dilution was discarded, replaced by PBST+ containing DAPI, and incubated for 15 min. Plates were rinsed once with PBST and washed twice for 10 min in PBS. All washing steps were done with 80 μl/well. Washing solution was added manually or using Perkin Elmer Janus (384 well Head, slowest dispensing speed).
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