At 28 days following the operation, the sciatic nerve tissues containing the area of crush injury site (5 mm away from the sciatic notch) were harvested and fixed with 4% paraformaldehyde. Then the longitudinal sections and transverse sections of the nerve tissue were prepared. Moreover, the sections were stained with NF200 (CST, 1:200), S100β (Abcam, 1:200), TuJ1 (Abcam, 1:200), MBP (Abcam, 1:200), CD31 (Abcam, 1:200), CD34 (Abcam, 1:200), VEGFR (Abcam, 1:200), GAPDH (Abcam, 1:200), Akt (Abcam, 1:500), p-AKT (Abcam, 1:500), PI3K (Abcam, 1:500), p-PI3K (ThermoFisher, 1:500) and PTEN (Abcam, 1:500). Secondary antibodies were as follows:Alexa Fluor568–conjugated Goat Anti-Rabbit IgG (Abcam), CoraLite594-conjugated Goat Anti-Mouse IgG (Proteintech, China), CoraLite488-conjugated Goat Anti-Rabbit IgG (Proteintech), CY3-labeled goat anti-rabbit (Servicebio) and AlexaFluor594-labeled goat anti-rabbit IgG (Abcam). Besides, we also used FITC-Tyramide (Servicebio) and CY3-Tyramide (Servicebio) to amplify fluorescence intensity. Nuclei were stained with DAPI, and the sections were observed under confocal laser scanning microscopy. The percentages of the markers positive areas were calculated by dividing integrated option density by selected region area, then multiplied by 100%. All parameters were measured using ImageJ.
Cy3 labeled goat anti rabbit
Manufactured by Wuhan Servicebio Technology
Sourced in China
CY3-labeled goat anti-rabbit is a secondary antibody used in various immunoassay techniques. It is produced by immunizing goats with rabbit immunoglobulins and then conjugating the antibodies with the fluorescent dye Cyanine 3 (CY3). This product can be used to detect and visualize rabbit primary antibodies in applications such as immunofluorescence microscopy, Western blotting, and flow cytometry.
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Lab products found in correlation
Fitc tyramide, by Wuhan Servicebio Technology (2 mentions)
Fluorescence microscope, by Olympus (2 mentions)
Glial fibrillary acidic protein (gfap), by Wuhan Servicebio Technology (2 mentions)
P pi3k, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Abcam (1 mentions)
P akt, by Abcam (1 mentions)
Coralite488 conjugated goat anti rabbit igg, by Proteintech (1 mentions)
Alexa fluor 568 conjugated goat anti rabbit igg, by Abcam (1 mentions)
Coralite594 conjugated goat anti mouse igg, by Proteintech (1 mentions)
Cy3 tyramide, by Wuhan Servicebio Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Wuhan Servicebio Technology (1 mentions)
Neuronal nuclei (neun), by Proteintech (1 mentions)
In situ cell death assay kit, by Roche (1 mentions)
Olyvia, by Olympus (1 mentions)
4 protocols using cy3 labeled goat anti rabbit
1
Nerve Injury Evaluation Using Immunostaining
2
Immunofluorescent Staining of Brain Tissue
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Tissue sections were prepared as described above. The sections were incubated at 4°C overnight with primary antibodies against the following proteins: NeuN (anti-rabbit, 1 : 50, Proteintech, Wuhan, China), GFAP (anti-rabbit, 1 : 1000, Servicebio, Wuhan, China), and Iba1 (anti-rabbit, 1 : 500, Servicebio, Wuhan, China). Then, the sections were incubated with CY3-labeled goat anti-rabbit (1 : 300, Servicebio, Wuhan, China) fluorescent secondary antibody at room temperature for 50 min. TUNEL staining was then carried out using an in situ cell death assay kit (Roche, Mannheim, Germany). Nuclei were counterstained using DAPI (Servicebio, Wuhan, China). The sections were observed under a fluorescence microscope (Olympus).
3
Immunohistochemical Analysis of Spinal Cord
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Under deep anesthesia, the rats were perfused with 0.9% saline through the heart and then with 4% paraformaldehyde. L4–L6 segmental spinal cord tissue was collected and fixed in 4% paraformaldehyde at 4°C overnight. Spinal tissue was continuously dehydrated in 20% and 30% sucrose and cut into 10-μm thick sections in a cryostat. The sections were blocked in 10% goat serum at room temperature for 2 h and incubated at 4°C overnight with primary antibodies against the following proteins: PKA (anti-mouse, 1 : 100, Santa Cruz, Dallas, TX, USA), neuronal nuclei (NeuN) (anti-rabbit, 1 : 50, Proteintech, Wuhan, China), glial fibrillary acidic protein (GFAP) (anti-rabbit, 1 : 500, Servicebio, Wuhan, China), and ionized calcium binding adapter molecule 1 (Iba1) (anti-rabbit, 1 : 500, Servicebio, Wuhan, China). Then, the sections were incubated with CY3-labeled goat anti-rabbit (1 : 300, Servicebio, Wuhan, China), 488-labeled goat anti-mouse (1 : 400, Servicebio, Wuhan, China) fluorescent secondary antibody, or a mixture of the two at room temperature for 1 h. The sections were washed three times with PBS and observed under a fluorescence microscope (Olympus, Tokyo, Japan), and images were acquired.
4
Immunofluorescence Analysis of Skin Lesions
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The skin lesion tissues were fixed, paraffin-embedded, sliced sections (thickness 3 μm), and dewaxed. Then, microwave oven antigen repair in citrate buffer, endogenous peroxidase was blocked by 3% hydrogen peroxide, serum was blocked. Next, add F4/80 antibody (1:100, cat no: 28463-1-AP, Proteintech, Wuhan) at 4℃ overnight, dropwise add HRP-labeled secondary antibody (1:100, cat no: GB23303, Servicebio, Wuhan) and incubate at room temperature for 30 min, add FITC-Tyramide (1:500, cat no: G1222, Servicebio, Wuhan) and incubate at room temperature for 10 min. Sections were submerged into citrate buffer microwaved for 10 min, followed by the addition of antibodies against iNOS (1:100, cat no: GB11119, Servicebio, Wuhan), CD206 (1:100, cat no: GB113497, Servicebio, Wuhan), and Notch1 (1:100, cat no: 11976R, Bioss, Beijing) incubated overnight at 4℃, and dropwise addition of fluorescent secondary antibody (CY3 labeled goat anti-rabbit, cat no: GB21303, Servicebio, Wuhan) was incubated at 37℃ for 30 min. Images were captured using digital scanning and browsing software (OlyVIA, OLYMPUS, Japan) and the fluorescence intensity of all acquired images was measured using the Image-J analysis system (National Institutes of Health, USA).
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