Immunoperoxidase staining was based on previously described protocols with modifications (Kardon, 1998 ) using an anti‐neurofilament antibody (Millipore AB1987) and a peroxidase conjugated secondary antibody (Sigma). Following overnight fixation in 4% PFA at 4˚C, embryos were washed in PBS and then placed in Dent's bleach overnight at 4˚C. Embryos were washed in methanol and fixed in Dent's fixative overnight at 4˚C. After being rinsed in PBS, embryos were blocked and then placed in the primary antibody for 2 days at 4˚C. Following multiple washes in PBS, embryos were placed in the secondary antibody overnight at 4˚C. Following PBS washes, embryos were incubated in fresh 3,3‐diaminobenzidine (DAB; Sigma) until reaction was complete, rinsed in methanol, and cleared and stored in BABB.
Anti neurofilament antibody
Manufactured by Merck Group
The Anti‐neurofilament antibody is a laboratory research tool used to detect and study neurofilament proteins, which are structural components of neurons. It can be used in various immunological techniques, such as Western blotting and immunohistochemistry, to identify and analyze neurofilament expression in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Complete neurobasal medium, by Thermo Fisher Scientific (2 mentions)
Poly d lysine hydrobromide, by Merck Group (2 mentions)
Glutamax 1, by Thermo Fisher Scientific (2 mentions)
Peroxidase conjugated secondary antibody, by Merck Group (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Nunc lab tek chamber slide system, by Thermo Fisher Scientific (1 mentions)
Nunc lab tek chamber slide, by Merck Group (1 mentions)
Serum free b 27, by Thermo Fisher Scientific (1 mentions)
Matrigel, by BD (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
4 protocols using anti neurofilament antibody
1
Immunoperoxidase Staining of Embryos
2
Whole-mount Immunofluorescence of Embryos
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Immunofluorescence staining was carried out based on previous protocols with some modifications (Kardon, 1998 ) using an anti‐neurofilament antibody (Millipore AB1987) and IgG goat anti‐rabbit Cy3 secondary antibody (Jackson ImmunoResearch). No antigen retrieval was carried out. Following overnight fixation in 4% PFA at 4˚C, embryos were washed in PBS and then placed in Dent's bleach (1 part hydrogen peroxide, 2 parts Dent's fix) overnight at 4˚C. Embryos were washed in 100% methanol and fixed in Dent's fixative (1 part Dimethyl sulfoxide, 4 parts methanol) overnight at 4˚C. After being rinsed multiple times in PBS, embryos were placed in the primary antibody in blocking solution overnight (18 hr minimum) at 4˚C. Following washes in PBS (over 6 hr), embryos were placed in the secondary antibody overnight at 4˚C. Embryos were then rinsed in PBS multiple times over a 6‐hr period, then placed in a 50% methanol:PBS solution, followed by 100% methanol washes, before being placed in BABB (33.3% benzyl alcohol, 66.6% benzyl benzoate) to clear and allow imaging under a fluorescence microscope.
3
Isolation and Culture of Mouse Neuronal Tissues
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Mouse MPG and DRG tissues were harvested and maintained as described previously [21 (link)22 (link)]. Briefly, MPG and DRG tissues were isolated from male mice under a dissecting microscope and transferred into sterile vials containing HBSS. After rinsing and washing twice with PBS, MPG and DRG tissues were cut into small pieces and plated onto a poly-D-lysine hydrobromide (0.1 mg/mL; Sigma-Aldrich)-coated 8-well Nunc Lab-Tek Chamber Slide System. MPG and DRG tissues were entirely covered with Matrigel, and plates were incubated for 10 to 15 minutes at 37°C in a 5% CO2 atmosphere. Then, 500 µL of complete Neurobasal medium (Gibco) Supplemented with 2% serum-free B-27 (Gibco) and 0.5 nM GlutaMAX-I (Gibco) were added and plates with MPG and DRG tissues were incubated in normal-glucose (5 mM) or high-glucose (30 mM) medium, with or without Hsp70 (500 ng/mL) for 5 days. Neurite outgrowth segments were then fixed in 4% paraformaldehyde for at least 30 minutes and immunostained with an anti-neurofilament antibody (Sigma-Aldrich; 1:50).
4
Culturing Murine Pelvic Ganglia
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MPG tissue was harvested and maintained as described previously.15 (link) Briefly, MPG tissue was isolated from male mice under a dissecting microscope and transferred into sterile vials containing Hank's Balanced Salt Solution (HBSS; Gibco). MPG tissue was then cut into small pieces and plated on 8-well Nunc Lab-Tek Chamber Slides coated with 0.1 mg/ml poly-D-lysine hydrobromide (Sigma-Aldrich). Tissue was covered with Matrigel (Ca# 354234, Becton Dickinson, Mountain View, CA, USA) and then plates were incubated for 5-10 minutes at 37°C in a humidified 5% CO2 atmosphere, after which 200 µl of complete Neurobasal medium (Ca# 21103-049, Gibco) supplemented with 2% serum-free B-27 (Ca# 17504-044, Gibco) and 0.5 nM GlutaMAX-I (Ca# 35050-061, Gibco) was added. To mimic the in vivo neuroinflammatory conditions of CNI-induced ED, we treated MPG tissue with 2.5 μg/ml LPS (Sigma-Aldrich) immediately after adding medium, as described previously.30 (link) The medium was changed every 2 days, and 5 days later, tissue was fixed in 4% paraformaldehyde for at least 30 minutes and neurite outgrowths were immunostained with an anti-neurofilament antibody (Ca# N5389, 1:50; Sigma-Aldrich).
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