Immunostaining was performed as described previously.11 (link) Briefly, 10-μm TA sections were blocked with 5% BSA and then incubated using antibodies against total α7 integrin, β1D Integrin, β-Dystroglycan, and Utrophin as described previously, followed by fluorescein isothiocyanate (FITC) donkey anti-rabbit (1:1,000, Jackson ImmunoResearch, Baltimore, MD) and mounting using Vectashield containing DAPI (H-1500, Vector Laboratories, CA). Images were taken as described previously. Capillary staining was performed using Alexa Fluor 488 anti-mouse CD31 (BioLegend, 102513).
Vectashield containing dapi h 1500
Manufactured by Vector Laboratories
Sourced in United States
Vectashield containing DAPI (H-1500) is a mounting medium designed to preserve fluorescence signals while counterstaining cell nuclei. It is a ready-to-use solution that can be used with a variety of fluorescent labels.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Ab11427, by Abcam (2 mentions)
C8035, by Merck Group (2 mentions)
Fluorescein isothiocyanate fitc donkey anti rabbit, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 488 donkey anti goat, by Thermo Fisher Scientific (1 mentions)
A21206, by Thermo Fisher Scientific (1 mentions)
Sc 991, by Santa Cruz Biotechnology (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
4 protocols using vectashield containing dapi h 1500
1
Immunostaining of Muscle Tissue Sections
2
Chondroitin Sulfate and Parvalbumin Labeling
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One well from each hemisphere of all birds was double-labeled for parvalbumin (PV) and chondroitin sulfate, one of the main components of the perineuronal nets, following a previously described protocol [22 (link), 37 (link), 41 (link)]. Briefly, sections were blocked in 5% Normal Goat Serum (NGS) diluted in Tris-buffered Saline (TBS) with 0.1% Triton-X-100 (TBST) for 30 minutes. They were incubated overnight at 4°C in a mixture of 2 primary antibodies diluted in TBST: a mouse monoclonal anti-chondroitin sulfate antibody (clone CS-56, 1:500 for Experiment 1 and 3 or 1:1000 for Experiment 2, C8035, Sigma Aldrich) specific for the glycosaminoglycan portion of the chondroitin sulfate proteoglycans that are the main components of the PNN and a polyclonal rabbit anti-parvalbumin antibody (1:1000; ab11427, Abcam). Sections were then incubated at room temperature in a mixture of secondary antibodies diluted in TBST. A goat anti-mouse IgG coupled with Alexa 488 (green, 1:100, Invitrogen) was used to visualize PNN staining and a goat anti-rabbit IgG coupled with Alexa 546 (red, 1:200, Invitrogen) was used to visualize PV cells. Finally, sections were mounted on slides using TBS with gelatin and coverslipped with Vectashield containing DAPI (H-1500, Vector laboratories) to confirm that PNN that were not surrounding PV-positive cells were localized around a cell nucleus.
3
Nurr1 Immunofluorescence Staining Protocol
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Coverslips were placed on the bottom of six-well plates and cells were plated as described earlier. After the exposures, the media were removed and the plates were washed 3× with PBS. Cells were fixed with 4% paraformaldehyde in PBS (MP Bio 199983) for 10 min. Cell plates were kept in PBS at 4°C until staining. Cells were permeabilized with ice-cold methanol for 2 min and blocked for 1 h with 10% rabbit serum and subsequently incubated in rabbit anti-Nurr1 (1 : 100) (SC 991; Santa Cruz Biotechnology) at 4°C overnight. After washing, the cells were incubated in AlexaFluor 488 donkey anti-goat for 1 h (A21206; Life Technologies, Carlsbad, California, USA). Coverslips were mounted onto slides with Vectashield containing DAPI (H-1500; Vector Laboratories Inc., Burlingame, California, USA).
4
Double-Labeling Chondroitin Sulfate and Parvalbumin in Avian Brains
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One well from each hemisphere of all birds was double-labeled for parvalbumin (PV) and chondroitin sulfate, one of the main components of the perineuronal nets, following a previously described protocol [22, 37, (link)41] (link). Briefly, sections were blocked in 5% Normal Goat Serum (NGS) diluted in Tris-buffered Saline (TBS) with 0.1% Triton-X-100 (TBST) for 30 minutes. They were incubated overnight at 4°C in a mixture of 2 primary antibodies diluted in TBST: a mouse monoclonal anti-chondroitin sulfate antibody (clone CS-56, 1:500 for Experiment 1 and 3 or 1:1000 for Experiment 2, C8035, Sigma Aldrich) specific for the glycosaminoglycan portion of the chondroitin sulfate proteoglycans that are the main components of the PNN and a polyclonal rabbit anti-parvalbumin antibody (1:1000; ab11427, Abcam). Sections were then incubated at room temperature in a mixture of secondary antibodies diluted in TBST. A goat anti-mouse IgG coupled with Alexa 488 (green, 1:100, Invitrogen) was used to visualize PNN staining and a goat anti-rabbit IgG coupled with Alexa 546 (red, 1:200, Invitrogen) was used to visualize PV cells. Finally, sections were mounted on slides using TBS with gelatin and coverslipped with Vectashield containing DAPI (H-1500, Vector laboratories) to confirm that PNN that were not surrounding PV-positive cells were localized around a cell nucleus.
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