A conventional immunohistochemistry protocol was performed on paraffin sections. Briefly, sections were deparaffinized in xylene followed by progressive rehydration with decreasing concentrations of ethanol. Antigen retrieval was performed by proteolytic enzyme digestion using Tris-EDTA Buffer (10 mM Tris; 1 mM EDTA, 20 mg ml−1 proteinase K, pH 8). Endogenous peroxidase was quenched using H2O2 30% in TBS 1×, before being blocked with 10% goat serum, 10 mg ml−1 BSA, 0.1% Triton X-100, TBS. Sections were incubated with avidin and biotin (Vector Laboratories), anti-CD34 antibody (catalog no. MA1-10202, Thermo Fisher Scientific) and horseradish peroxidase-coupled secondary antibody (catalog no. 115-035-006, The Jackson Laboratory). Samples were blocked with the ABC Kit (Vector Laboratories) and incubated with a substrate of peroxidase, 3,3′-diaminobenzidine (SIGMAFAST, Sigma-Aldrich). Sections were stained in hematoxylin, rinsed in water and dipped in 1% acid alcohol, and then mounted with aqueous mounting medium (Vector Laboratories). Slides were imaged using a slide scanning microscope (ZEISS Axioscan) and analyzed manually to measure the distance and areas, and quantify cells and vessels using the NDP2.view software (Hamamatsu).
Anti cd34 antibody
Manufactured by Thermo Fisher Scientific
The Anti-CD34 antibody is a laboratory reagent used for the detection and identification of CD34-positive cells. CD34 is a surface glycoprotein expressed on hematopoietic stem and progenitor cells. This antibody can be used in various immunoassay techniques to study the presence and distribution of CD34-positive cells in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700, by Zeiss (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Aqueous mounting medium, by Vector Laboratories (1 mentions)
Horseradish peroxidase coupled secondary antibody, by Jackson ImmunoResearch (1 mentions)
Avidin and biotin, by Vector Laboratories (1 mentions)
Ndp2 view software, by Hamamatsu Photonics (1 mentions)
Abc kit, by Vector Laboratories (1 mentions)
Axioscan, by Zeiss (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Meyer s hematoxylin solution, by Fujifilm (1 mentions)
Triton x 100, by Thermo Fisher Scientific (1 mentions)
Oct compound, by Sakura Finetek (1 mentions)
Anti p akt ser473 antibody, by Cell Signaling Technology (1 mentions)
Anti p s6rp antibody, by Cell Signaling Technology (1 mentions)
Anti cd31 antibody, by Dianova (1 mentions)
Dp70 camera, by Olympus (1 mentions)
4 protocols using anti cd34 antibody
1
Immunohistochemical Analysis of CD34 Expression
2
Immunohistochemical Analysis of CD34+ Cells
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Samples were washed with PBS and fixed with 10% formaldehyde (FUJIFILM-Wako
Pure Chemical Corporation), overnight at 25 °C. After embedding
in optimal cutting temperature compound (O.C.T. compound; Sakura Finetek,
Japan), 8 μm thick sections were cut and stained with Meyer’s
hematoxylin solution (FUJIFILM-Wako Pure Chemical Corporation) and
1% eosin Y solution (Muto Pure Chemical, Tokyo, Japan). For immunohistochemical
staining, the sections were fixed in 4% formaldehyde for 1 h at 25
°C. The samples were first blocked in PBS containing 1% bovine
serum albumin (Thermo Fisher Scientific, Inc.) and 0.01% Triton X-100
(Thermo Fisher Scientific, Inc.) for 1 h at 25 °C and subsequently
incubated overnight with anti-CD34 antibody (Thermo Fisher Scientific,
Inc.) at 4 °C. The sections were incubated with the corresponding
secondary antibodies in blocking solution for 3 h at 25 °C and
finally with DAPI in PBS for 10 min. A confocal microscope (LSM 700;
Carl Zeiss) was used for fluorescence imaging.
Pure Chemical Corporation), overnight at 25 °C. After embedding
in optimal cutting temperature compound (O.C.T. compound; Sakura Finetek,
Japan), 8 μm thick sections were cut and stained with Meyer’s
hematoxylin solution (FUJIFILM-Wako Pure Chemical Corporation) and
1% eosin Y solution (Muto Pure Chemical, Tokyo, Japan). For immunohistochemical
staining, the sections were fixed in 4% formaldehyde for 1 h at 25
°C. The samples were first blocked in PBS containing 1% bovine
serum albumin (Thermo Fisher Scientific, Inc.) and 0.01% Triton X-100
(Thermo Fisher Scientific, Inc.) for 1 h at 25 °C and subsequently
incubated overnight with anti-CD34 antibody (Thermo Fisher Scientific,
Inc.) at 4 °C. The sections were incubated with the corresponding
secondary antibodies in blocking solution for 3 h at 25 °C and
finally with DAPI in PBS for 10 min. A confocal microscope (LSM 700;
Carl Zeiss) was used for fluorescence imaging.
3
Immunostaining of Kidney Sections
4
Evaluating Axonal Regeneration and Angiogenesis
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The nerve conduit and grafted nerves were harvested at 12 weeks post-surgery, and immersed in 4% paraformaldehyde overnight at 4 °C. The specimens were embedded in paraffin and five-micrometer-thick central transverse sections were examined by immunohistochemistry. To evaluate the regenerating axons at the central transverse section, the neurofilament antibody-positive axons (anti-neurofilament protein antibodies (1:100; DAKO, Glostrup, Denmark)) were analyzed using computer-assisted imaging. An image of the transverse section, in which the greatest number of regenerating axons was found, was photographed at a magnification of ×400 with an Olympus DP70 camera (Olympus, Tokyo, Japan). The number of the neurofilament antibody-positive axons were counted automatically using ImageJ software, wherein the axons were stained clearly (National Institutes of Health, http:// imagej.nih.gov/ij/). Also, to examine angiogenesis, the specimen of the central transverse section in the nerve conduits with bFGF gelatin group and nerve conduits with SDF-1/bFGF gelatin group were immunohistochemically stained using anti-CD34 antibody (1:200; eBioscience, San Diego, CA).
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