At specified time points after nerve grafting, the artificial nerve bridging effects were evaluated by observing the graft appearance. Grafts were frozen (-80°C), sectioned (6 µm thickness) using a cryostat (CM1850, LEICA, Germany), stained using immunofluorescent markers or HE, and examined under a laser scanning confocal microscope (LSM200, Zeiss, Germany) to identify different cell types and fibroin fibers within the graft. Nuclei were stained blue using 4’,6-diamidino-2-phenylindole (Biosynthesis Biotechnology, China). Primary antibody for low-affinity nerve growth factor receptor (NGFR) P75 (goat anti-rat; Santa Cruz Biotechnology, USA) was used to identify Schwann cells. Primary antibodies against fibronectin (goat anti-rat; Santa Cruz Biotechnology) and NF200 (rabbit anti-rat; Sigma, USA) were used to identify fibroblasts and axons, respectively. Schwann cells and fibroblasts were labelled green using donkey anti-goat secondary antibody and axons were marked red using donkey anti-rabbit secondary antibody (Life Technologies, USA). Integrin-α(rabbit anti-rat; Santa Cruz Biotechnology, USA ) were used to identify inflammatory cells. Specific steps for immunofluorescence staining were performed according to the manufacturer’s instructions. Some of the grafts were observed with a scanning electron microscope (SEM) (JSM-6510LV, Japan).
Donkey anti rabbit secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Donkey anti-Rabbit secondary antibody is a laboratory reagent used in immunoassays and other immunological techniques. It is designed to bind to Rabbit primary antibodies, allowing for the detection and visualization of target antigens. The antibody is derived from donkeys and is specific to Rabbit IgG.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti fibrinogen antibody, by Abcam (3 mentions)
Cm1850, by Leica (2 mentions)
Nf200, by Merck Group (2 mentions)
Lsm200, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Bio-Synthesis (2 mentions)
Primary antibodies against fibronectin, by Santa Cruz Biotechnology (2 mentions)
Donkey anti goat secondary antibody, by Thermo Fisher Scientific (2 mentions)
Fv1200 confocal microscope, by Olympus (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (2 mentions)
Cy3 donkey anti rabbit, by Thermo Fisher Scientific (2 mentions)
Protein block serum free, by Agilent Technologies (1 mentions)
Ab6640, by Abcam (1 mentions)
Ab485606, by Abcam (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Tris buffered saline (tbs), by Beyotime (1 mentions)
Bx53 microscope, by Olympus (1 mentions)
Lc3 primary antibody, by Abcam (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Axio vert a1 fluorescence microscope, by Zeiss (1 mentions)
18 protocols using donkey anti rabbit secondary antibody
1
Nerve Graft Evaluation via Immunofluorescence
2
Nanoparticle Tissue Distribution and Oxidative Injury
3
Schwann Cell and Axon Identification in Nerve Grafts
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At specified time points after nerve grafting, the artificial nerve bridging effects were evaluated by observing the graft appearance. Grafts were frozen (–80°C), sectioned (6 µm thickness) using a cryostat (CM1850, LEICA, Germany), stained using immunofluorescent markers, and examined under a laser scanning confocal microscope (LSM200, Zeiss, Germany) to identify different cell types within the graft. Nuclei were stained blue using 4′,6‐diamidino‐2‐phenylindole (Biosynthesis Biotechnology, China). Primary antibody for low‐affinity nerve growth factor receptor (NGFR) P75 (goat anti‐rat; Santa Cruz Biotechnology, USA) was used to identify Schwann cells. Primary antibodies against fibronectin (goat anti‐rat; Santa Cruz Biotechnology) and NF200 (rabbit anti‐rat; Sigma, USA) were used to identify fibroblasts and axons, respectively. Schwann cells and fibroblasts were labeled green using donkey anti‐goat secondary antibody and axons were marked red using donkey anti‐rabbit secondary antibody (Life Technologies, USA). Specific steps for immunofluorescence staining were performed according to the manufacturer's instructions.
4
Immunofluorescence Assay of γδT Cells
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Immunofluorescence assays of the purified peripheral γδT cells were carried out by Biossci Company. Blocking was performed in Tris‐buffered saline (Beyotime) with 10% normal serum (Biological Industries) and 0.1% Triton X‐100 (Beyotime). The 1:50 diluted rabbit anti‐human CD206 antibody (Abcam) and the 1:400 diluted donkey anti‐rabbit secondary antibody (Life Technologies) were used for the immunofluorescence assay. DAPI dye (Gibco) was used at room temperature for 20 min at a 1:500 dilution to identify the nucleus. The images were aquired on an Olympus BX53 microscope.
5
Immunofluorescence Imaging of LC3
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The tissue sections undergo dewaxing, rehydration, and antigen repair extraction. Then, LC3 primary antibody (1 : 200 dilution, Abcam) was used for tissue sections to be incubated at 4°C overnight, donkey anti-rabbit secondary antibody (1 : 200 dilution, Life Technologies) was incubated at 37°C for 45 min, the cell nucleus was stained with DAPI dye, and the tablet was sealed with fluorescent tablet. Photo by fluorescence microscope (Olympus).
6
Visualizing Nrf2 Expression in BEAS-2B Cells
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The expression of nuclear factor erythroid-2-related factor 2 (Nrf2) in BEAS-2B cells was analysed using immunofluorescence staining. Complete BEBM was removed and the cells were washed using phosphate-buffered saline (PBS). The cells were subsequently fixed using ice cold 4% paraformaldehyde for 15 min at room temperature. The cells were washed 3 times in PBS for 5 min each. The cells were then blocked using Blocking Buffer (1x PBS/5% normal goat serum; cat. no. ab7481; Abcam)/0.3% Triton X-100 (cat. no. T8787; Sigma Aldrich, Merck KGaA;) for 60 min at room temperature. The cells were washed again and incubated with a primary antibody against Nrf2 (Sigma-Aldrich, Merck KGaA; cat. no. SAB4501984) overnight at 4˚C. The following day, the cells were washed 4 times, 10 min each time, with PBS and incubated with a donkey-anti-rabbit secondary antibody (Thermo Fisher Scientific, Inc.; cat. no. A-21207; 1:200) for 1.5 h at room temperature. The cells were subsequently counterstained with the nuclear stain DAPI (Sigma-Aldrich, Merck KGaA; 0.6 mM in PBS) for 4 min at room temperature. Stained cells were imaged using a Zeiss AxioVert A1 fluorescence microscope (Carl Zeiss AG; x40 magnification). A total of 6 fields were analysed.
7
Immunohistochemical Analysis of Temporal Bone
8
Immunohistochemical Analysis of Retinal Ganglion Cells
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Mouse eyes were fixed with 4% PFA and then were transferred to PBS. The retinas were removed, permeabilized with 0.5% Triton X-100, blocked with 0.5% Triton X-100 containing 10% donkey serum, and then incubated overnight in 0.2% Triton X-100/10% donkey serum in PBS containing the anti-beta III Tubulin antibody (anti-Tubb3; 802,001, BioLegend, USA). The next day, a donkey anti-rabbit secondary antibody (ThermoFisher Scientific, USA) in 0.15 % Tween 20/PBS was applied for 1.5 h at room temperature. After washing with 0.15 % Tween 20/PBS, retinas were flat-mounted (RGC layer facing up), cover-slipped, and imaged with a Leica STELLARIS confocal microscope (Leica Microsystems, USA). Tubb3-positive RGCs were imaged randomly to collect images from four retinal quadrants and were counted with ImageJ software (version 1.53o; https://imagej.nih.gov/ ; accessed on 11 January 2022 to download ImageJ). RGC survival in the IR retinas was calculated as a percentage of the mean cell density in normotensive fellow control eyes.
9
Evaluating Cardiomyocyte Apoptosis via EV Treatment
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Primary cardiomyocytes were treated with 10µg of EVs per 4x10 5 cells for 24 hours prior to fixation with 4% PFA for 15 minutes and permeabilization for 20 minutes at room temperature.
Subsequent TUNEL assay for apoptosis was performed using Click-iT TUNEL Alexa Fluor 488
Imaging Assay (Thermo Fisher) following manufacturer's protocol. Cells were then incubated with Cardiac TnI primary antibody (Rabbit polyclonal, Proteintech) at 1:100 dilution overnight at 4 o C.
Donkey anti-rabbit secondary antibody (Thermo Fisher) was incubated with cells at a 1:400 dilution for 1 hour at room temperature in the dark. DAPI (1X) was incubated with cells for 5 minutes for nuclear staining followed by PBS washing and imaging. Images were obtained using an EVOS FL Auto Imaging System. Apoptosis was assessed as the percent of TUNEL positive cardiomyocytes.
Subsequent TUNEL assay for apoptosis was performed using Click-iT TUNEL Alexa Fluor 488
Imaging Assay (Thermo Fisher) following manufacturer's protocol. Cells were then incubated with Cardiac TnI primary antibody (Rabbit polyclonal, Proteintech) at 1:100 dilution overnight at 4 o C.
Donkey anti-rabbit secondary antibody (Thermo Fisher) was incubated with cells at a 1:400 dilution for 1 hour at room temperature in the dark. DAPI (1X) was incubated with cells for 5 minutes for nuclear staining followed by PBS washing and imaging. Images were obtained using an EVOS FL Auto Imaging System. Apoptosis was assessed as the percent of TUNEL positive cardiomyocytes.
10
Fibrinogen Quantification in Post-SAH Microvessels
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As per our published methods [10 (link)], ipsilateral (left) cortical microvessel thrombosis was evaluated for fibrinogen by immunofluorescence staining seventy-two hours post SAH. Briefly, free floating, fixed brain sections with 50-μm thickness were incubated overnight at 4 °C with primary rabbit anti-fibrinogen antibody (1:3000, Abcam, Cambridge, MA, USA), followed by overnight incubation at 4 °C with donkey anti-rabbit secondary antibody (1:2000, Invitrogen Waltham, MA, USA). Brain sections were then analyzed for fibrinogen using a NanoZoomer 2.0-HT digital slide scanner (Hamamatsu Corporation, Bridgewater, NJ, USA), followed by estimation of microvessel thrombosis density using Image J (v.1.53t).
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