The experiment was performed as described previously.24 (link) Briefly, whole-mount limbal tissues were harvested at day 7 post-procedure, fixed in 4% paraformaldehyde, and immunostained with primary rat-anti-mouse CD31 antibody (BD Pharmingen, San Diego, CA, USA) that was visualized by Cy3-conjugated donkey anti-rat secondary antibody (Jackson-Immuno Research Laboratories, West Grove, PA, USA). Samples were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) and examined by an AxioImager M1 epifluorescence deconvolution microscope with AxioVision 4.8 software (Carl Zeiss AG, Göttingen, Germany).
Cy3 conjugated donkey anti rat secondary antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States, United Kingdom
Cy3-conjugated donkey anti-rat secondary antibody is a laboratory reagent that binds to primary antibodies raised in rat. The Cy3 fluorescent dye is conjugated to the secondary antibody, enabling detection and visualization of the target antigen.
Automatically generated - may contain errors
Lab products found in correlation
Axiovision 4, by Zeiss (1 mentions)
Primary rat anti mouse cd31 anti body, by BD (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Cfi super plan apo 20 objective, by Nikon (1 mentions)
Rat anti rfp, by Proteintech (1 mentions)
Alexa 647 conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
C2 confocal microscope, by Nikon (1 mentions)
Confocal laser scanning microscope, by Leica (1 mentions)
Anti timp 1, by Cell Signaling Technology (1 mentions)
Rat anti ki 67, by Abcam (1 mentions)
Anti α tubulin, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Abcam (1 mentions)
Trichrome stain masson kit, by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
5 protocols using cy3 conjugated donkey anti rat secondary antibody
1
Whole-mount Limbal Tissue Immunostaining
2
Lymphatic Vessel Imaging in Popliteal Lymph Nodes
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At 2 h after Cy5.5-HA10K injection, popliteal LNs were harvested and were sectioned into 10 μm slices by using an Ultrapro 5000 cryostat (Vibratome). Slices were fixed with Z-fix solution for 15 min and then were blocked with PBS containing 1% BSA for 1 h. Slices were incubated with rat anti-mouse LYVE-1 primary antibody (1:400, MBL International Corporation) at room temperature for 2 h, and then with Cy3-conjugated donkey anti-rat secondary antibody (1:200; Jackson ImmunoResearch Laboratories). Between each step, the slices were gently washed 5 times with 0.05% tween 20 PBS solution (PBST) for 5 min each time. The slices were then incubated with medium containing 4′,6-diamidino-2-phenylindole (DAPI). The staining observation was done with an epifluorescence microscope (X81; Olympus). H&E staining was performed as previously reported 27 (link).
3
Immunohistochemical Visualization of Recorded Neurons
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After in vitro recordings slices were fixed in 4% paraformaldehyde overnight at 4°C and biocytin content of the recorded neurons was visualized using Alexa 647-conjugated streptavidin (1:10,000 or 1:20,000, Invitrogen). mCherry and GFP or EYFP signals in the neurons were enhanced by immunostainings using the following antibodies: rat anti-RFP (1:1000, Chromotek) revealed by Cy3-conjugated donkey anti-rat secondary antibody (1:500, The Jackson Laboratory) and chicken anti-GFP (1:1000, SYSY) revealed by Alexa 488-conjugated donkey anti-chicken antibody (1:500, The Jackson Laboratory). Slices were mounted in Vectashield (Vector Laboratories) and 3D images were obtained using a Nikon C2 confocal microscope with Nikon CFI Super Plan Apo 20× objective (N.A. 0.75; z step size: 1 μm, xy: 0.31 μm/pixel). Images were processed and analyzed with the NIS Elements AR 5.30.01 software (Nikon Instruments).
4
Immunohistochemical Identification of Patched Cells
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For post hoc identification of the patched cells, slices were fixed in 4% paraformaldehyde (PFA) in phosphate buffer (PB) for 12–24 h and then stored in anti-freeze solution (ethylenglycol and glycerol in PB buffer) at −20°C until processed. For immunohistochemical staining against, mCherry/tdTomato and biocytin, slices were rinsed three times with KPBS and pre-incubated for 1 h in blocking solution (10% normal donkey serum and 0.25% Triton X-100 in KPBS, T-KPBS). The sections were then incubated overnight with 1:1000 rat anti-mRFP (5F8, Chromotek, Germany) in 5% serum blocking solution, rinsed three additional times in T-KPBS and incubated for 2 h in Cy3-conjugated donkey anti-rat secondary antibody (1:400, Jackson Immunoresearch, Suffolk, UK) and Alexa 488-conjugated streptavidin-D (1:200, Molecular Probes) in 5% serum blocking solution. Slices were finally rinsed three times in KPBS, mounted on coated slides and cover-slipped with DABCO.
For reconstruction of the axonal arborization of PV-tdTomato-positive neurons, labeled cells were examined with a confocal laser-scanning microscope (Leica). Confocal Z-stacks were obtained along the entire dendritic and axonal tree of the cells.
For reconstruction of the axonal arborization of PV-tdTomato-positive neurons, labeled cells were examined with a confocal laser-scanning microscope (Leica). Confocal Z-stacks were obtained along the entire dendritic and axonal tree of the cells.
5
Protein Extraction and Analysis Techniques
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Protein was extracted from the cultured cells or conditioned media by RIPA buffer (Sigma-Aldrich) for Western Blot. Primary antibodies for Western Blotting are anti-TIMP-1 and anti-a-tubulin (Cell Signaling, San Jose, CA). The secondary antibody is HRP-conjugated anti-rabbit (Jackson Labs, Bar Harbor, ME). Images shown in the figure were representative from five repeats. Densitometry of Western blots was quantified with NIH ImageJ software (Bethesda, MA). Massontrichrome staining was performed using a Trichrome Stain (Masson) Kit (Sigma-Aldrich). For immunohistochemistry, the primary antibody is rat anti-Ki-67 (Abcam, Cambridge, MA) and anti-TIMP-1 (Cell Signalling). Indirect fluorescent staining was performed with Cy3-conjugated donkey anti-rat secondary antibody (Jackson Labs). Nuclear staining was performed with DAPI (Abcam). Quantification of at least 500 cells was done in 5 repeats in each condition.
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