Immunostaining was performed as previously described14 (link). Sections were fixed with 4% paraformaldehyde in 20 mM Tris-HCl (pH 7.4) containing 150 mM NaCl, 3 mM KCl, and 0.1% Tween 20 (TBS-T) for 20 min, washed with TBS-T, and then incubated with 0.25% Triton X-100 in TBS-T for 15 min. The sections were treated with 10 mM citrate solution (pH 6.0) for antigen retrieval at 95 °C for 15 min, washed with TBS-T, and then blocked in 10% goat serum (Sigma-Aldrich, St. Louis, MO) in TBS-T for 1 h. Sections were incubated with primary antibody (MBP, 1:500 BioLegend, San Diego, CA; Olig2, 1:500 Abcam, cambridge UK; NeuN, 1:500 MBL, Nagoya, JAPAN; GFAP, 1:500 Cell Signaling Technology, Beverly MA; Iba1, 1:500 Wako, Japan) with 10% goat serum in TBS-T at 4 °C overnight, washed with TBS-T, and then incubated with CFTM 594 goat anti-rabbit IgG(H + L) (1:1000 Biotium, Hayward, CA) and CFTM 488 goat anti-mouse IgG (H + L) (1:1000 Biotium) at room temperature for 2 h. After being washed, the sections were mounted using Fluoromount (Diagnostic BioSystems, Pleasanton, CA).
Goat serum
Manufactured by Biotium
Sourced in Japan
Goat serum is a cell culture reagent derived from the blood of healthy goats. It is a complex mixture of proteins, growth factors, and other biomolecules that can be used to supplement cell culture media and support the growth and maintenance of various cell types in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Goat serum, by Merck Group (2 mentions)
Olig2, by Abcam (1 mentions)
Glial fibrillary acidic protein (gfap), by Cell Signaling Technology (1 mentions)
Fluoromount, by Diagnostic BioSystems (1 mentions)
Prolong diamond, by Thermo Fisher Scientific (1 mentions)
P36970, by Thermo Fisher Scientific (1 mentions)
A27039, by Thermo Fisher Scientific (1 mentions)
A1 confocal microscope, by Nikon (1 mentions)
2 protocols using goat serum
1
Immunostaining of Brain Tissue Sections
2
Quantification of p62 Positive Fibers in TA
Check if the same lab product or an alternative is used in the 5 most similar protocols
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TA muscles were mounted in OCT, snap frozen, and sectioned in a cryotome at 10 µm thickness. Tissue sections were re-hydrated with three washes of 1X PBS of 5 minutes each. Sections were treated with 0.2% Triton X-100 in 1X PBS for 5 minutes followed by three washes of 1X PBS. Sections were blocked with 0.1% goat serum (Millipore Sigma#G0923) for 1 hour at room temperature followed by overnight primary antibodies incubation (SQSTM1/p62 Rabbit mAb #23214) at 1:200 dilution in 0.1 % goat serum in 4°C. Primary antibodies were washed 3 times with 1X PBS for 5 minutes each followed by incubation with goat anti-rabbit Alexa Fluor 555 (Invitrogen# A27039) 1:300 dilution in 0.1 % goat serum), WGA 488 (100 µg/ml, Biotium #29022), and DAPI at 2 µg/ml for 1 hour at room temperature. Slides were then mounted with Prolong Diamond and coverslipped (Invitrogen #P36970). Whole section images were taken using a Nikon A1 confocal microscope. FIJI was used to quantify the total number of p62positive fibers and minimum Feret diameter.
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