The epitope retrieval protocol for immunofluorescence was the same as that for immunohistochemistry. Prior to primary antibody incubation, samples were blocked with normal horse serum (3%, Vector) followed by streptavidin and biotin blocking solutions (Vector) for 15 min each. Tissues were then blocked with Serum-Free Protein Block (Agilent) for 30 min and incubated in a primary antibody cocktail solution for 60 min. The following antibodies were used for immunofluorescence: CD4 (1:500, Abcam), PD1 (1:100, BioLegend), IgD (1:500, SouthernBiotech), and CD23 (1:750, Proteintech). Samples were rinsed with Immuno Wash prior to incubation in a cocktail mixture of horse host secondary antibodies for 30 min. The secondary antibodies include 1:300 Alexa Fluor 488 horse anti-rabbit (1:300, Vector), Alexa Fluor 594 horse anti-mouse (1:300, Vector), and biotinylated horse anti-goat (1:200, Vector). Tissues were then incubated in Alexa Fluor 680 Streptavidin (1:500, Thermo Fisher) for 30 min. Slides were counterstained with DAPI (Thermo Fisher Scientific) at 1 μg/mL in 1x PBS for 2 min.
Biotinylated horse anti goat
Manufactured by Vector Laboratories
Sourced in United Kingdom, Canada
Biotinylated horse anti-goat is a secondary antibody used in various immunoassay techniques. It is produced by immunizing horses with goat immunoglobulins and labeling the resulting antibodies with biotin.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin alexa568, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Normal horse serum, by Vector Laboratories (1 mentions)
Protein block serum free, by Agilent Technologies (1 mentions)
Alexa fluor 594 horse anti mouse, by Vector Laboratories (1 mentions)
Goat anti albumin, by Abcam (1 mentions)
Abc reagent, by Vector Laboratories (1 mentions)
Nanozoomer slide scanner, by Hamamatsu Photonics (1 mentions)
Axio imager z1 microscope, by Zeiss (1 mentions)
Ab18723, by Abcam (1 mentions)
Normal horse serum, by Jackson ImmunoResearch (1 mentions)
3 3 diaminobenzidine tetrahydrochloride dab, by Vector Laboratories (1 mentions)
Rabbit polyclonal igg, by Abcam (1 mentions)
Goat polyclonal igg, by Santa Cruz Biotechnology (1 mentions)
Sc 8066, by Abcam (1 mentions)
Biotinylated goat anti rabbit secondary antibody, by Vector Laboratories (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Krystalon, by Merck Group (1 mentions)
Avidin biotin complex, by Vector Laboratories (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
8 protocols using biotinylated horse anti goat
1
Immunofluorescence Staining of Immune Cells
2
Cerebral Albumin Permeability in Hypoxic-Ischemic Injury
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Blood–brain barrier permeability to endogenous albumin (~66,000Da) was determined immunohistochemically at 2, 6, 24 hours and 3 days after HI along with P9 naïve controls. This abundant plasma protein is unlikely to be synthesized within the brain making it a suitable endogenous larger-sized permeability marker. Paraffin sections of the brain at the level of the hippocampus were deparaffinized and blocked as below and incubated with goat anti-albumin (1:6,000; Abcam, Cambridge, UK, Cat# ab19194) overnight followed by biotinylated horse anti-goat (1:250; Vector, Peterborough, UK) and further processed as below.
3
Dorsal Column Fiber Regeneration Analysis
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Dorsal hemisection was performed, as described (Liz et al. 2014 (link)) using a micro ophthalmic scalpel (Feather, Safety Razor Co. Ltd) in Thy1-cre+Kiaa0319+/+ and Thy1-cre+Kiaa0319F/F mice (n = 8 mice/group). Animals recovered for 5 weeks and 4 days prior to euthanasia, 2 μL of 1% cholera toxin-B (List Biologicals, Campbell, CA, USA) were injected in the left sciatic nerve. Serial spinal cord sagittal sections were collected for free floating immunohistochemistry with anti-cholera toxin-B (CT-B) (1:30 000; List Biologicals). Antigen detection was performed with biotinylated horse anti-goat (1:200; Vector) and streptavidin Alexa 568 (1:1000, Invitrogen). Image acquisition was done with a Laser Scanning Confocal Microscope (Leica SP5) and image analysis was performed with Fiji. Regeneration of dorsal column fibers was quantified by counting the total number of CT-B+/YFP+ axons within the glial scar. The length of the longest CT-B+/YFP+ axon found rostrally to the injury site was measured using as the origin a vertical line placed at the rostral end of the dorsal column tract.
4
Visualizing Neuronal Projections in Brain Stem and Spinal Cord
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Brain stems were sectioned transversely at 50μm on a cryostat and free floating sections were blocked with 2% horse serum with 0.1% triton X100, and incubated for 72 hours with goat anti-CTB (1:80.000; 703, List Laboratories Inc., Campbell, CA), followed by biotinylated horse anti-goat (1:300; Vector Labs), ABC reagent (1:200; Vector Labs), with washes in 1xTBS between each step, and finally developed using diaminobenzidine with nickel ammonium sulphate. Sections were then mounted onto Superfrost Plus slides (Menzel-Gläser), dehydrated and embedded with Entellan. Spinal cords were sectioned parasagittally at 20μm on a cryostat onto Superfrost Plus slides (Menzel-Gläser), and stained with the same procedure.
5
Proliferation Assay in Mouse Gastrointestinal Tissues
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For proliferation assays, mice were injected intraperitoneally with 5-bromo-2’-deoxyuridine (BrdU, 120 mg·kg−1) and 5-fluoro-2’-deoxyuridine (12 mg·kg−1) in sterile water 90 min before killing. Following killing, stomachs were immediately excised and flushed with phosphate-buffered saline then inflated with freshly prepared formalin (10% formaldehyde, Sigma, St. Louis, MO, USA) in phosphate-buffered saline and the pylorus clamped with a hemostat. Inflated stomachs and segments of the small and large intestines were allowed to fix overnight in 10% formalin then transferred to 70% ethanol. Tissues were arranged in 3% agar in a tissue cassette, underwent routine paraffin processing, and 5 μm sections were cut and mounted on glass slides. For immunohistochemistry, sections underwent a standard deparaffinization and rehydration protocol then were blocked with 5% horse serum for 1 h before staining for BrdU using Goat anti-BrdU (1:20 000, gift of Dr Jeff Gordon, Washington University) and biotinylated horse anti-goat (1:200, Vector Laboratories) antibodies. Images were acquired using a Nanozoomer Slide Scanner (Hamamatsu, Japan, model 2.0-HT).
6
Sciatic Nerve Crush Injury Model
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Sciatic nerve crush at the mid-thigh level was performed using Pean forceps, closed completely twice during 15s in Thy1-cre+Kiaa0319+/+ and Thy1-cre+Kiaa0319F/F mice (n = 5 mice/group). Animals recovered for 3 days, after which collection and sectioning of sciatic nerves at 20 μm was performed. Consecutive longitudinal sections were collected for free-floating immunofluorescence with sheep anti-GAP-43 (1:5000; kindly provided by Dr Larry Benowitz, Harvard Medical School) and antigen detection was performed following incubation with biotinylated horse anti-goat (1:100; Vector) and streptavidin Alexa 568 (1:1000, Invitrogen). Image acquisition was done with Zeiss Axio Imager Z1 microscope (using the same settings for all the samples analyzed) and image analysis was performed with ImageJ. The lesion site was determined as the area with severely decreased YFP staining. GAP-43 staining intensity was determined distally from the proximal border of the injury site.
7
Immunohistochemical Detection of DCX
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Sections were brought to room temperature in tris buffered saline (TBS), followed by a 10-min wash in fresh TBS. Sections were then incubated for 30-min in a H₂O₂ solution (97% TBS, 1% methanol, 2% of 3% retail H₂O₂) to eliminate endogenous peroxidases. After three 5-min TBS rinses, non-specific binding was blocked with 5% normal horse serum (Jackson ImmunoResearch Laboratories Inc.) and 0.5% Triton X-100 (Sigma-Aldrich) in TBS for 30-min at room temperature. This was followed by exposure to anti-DCX antibody (goat polyclonal IgG, Santa Cruz Biotechnology; sc-8066, 1:150; or rabbit polyclonal IgG, Abcam; ab18723, 1:1000) in the same blocking buffer overnight at 4˚C. After three 5-min TBS rinses, sections were incubated for 3 h in biotinylated horse anti-goat (1:200, Vector Labs) or biotinylated goat anti-rabbit secondary antibody (1:200, Vector Labs) in TBS, rinsed again, and exposed for 1 h to an avidin-biotin complex (Vector Labs). Sections were rinsed in TBS and then reacted in a solution of 0.04% of 3,3’-diaminobenzidine tetrahydrochloride (DAB, Vector Labs) with nickel until the tissue changed color (~3–10 min). Following three 5-min TBS rinses, sections were dehydrated in ethanols, delipidized in xylenes, and cover slipped with Krystalon (Millipore-Sigma).
8
Paraffin Embedding and IHC Protocol
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