Total protein was prepared using mouse brain homogenate, and the protein concentration was determined using a BCA Protein Assay Kit (Sigma). Proteins were separated by SDS‐PAGE on a 4%–20% Criterion TGX Stain‐Free Precast gels (Bio‐Rad). After blotting, the membranes were imaged for total protein. The membranes were incubated with anti‐OPN antibody (O‐17, IBL, 1:1000), anti‐MBP antibody (SMI94, BioLegend, 1:1000), or anti‐PLP1 antibody (ab9311, Abcam, 1:1000) followed by HRP‐conjugated secondary antibodies (Vector, 1:5000). Enhanced chemiluminescence was performed using the SuperSignal West Dura Extended Duration Substrate (Thermo Scientific), and protein expression levels were quantified using Image Lab (Bio‐Rad) and normalized against total protein levels.
Smi94
Manufactured by BioLegend
The SMI94 is a multipurpose laboratory instrument designed for various scientific applications. It features adjustable temperature control and stirring capabilities to facilitate efficient sample processing and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by Vector Laboratories (1 mentions)
Criterion tgx stain free precast gel, by Bio-Rad (1 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Image lab, by Bio-Rad (1 mentions)
Alexa fluor 594 and 488 conjugated secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Ab9610, by BioLegend (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Fluorsave, by Merck Group (1 mentions)
Smi31, by BioLegend (1 mentions)
Smi 32, by BioLegend (1 mentions)
Glial fibrillary acidic protein (gfap), by Agilent Technologies (1 mentions)
4 protocols using smi94
1
Quantitative Protein Analysis in Mouse Brain
2
Oligodendrocyte Quantification and Myelination Assay
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Oligodendrocyte cultures were fixated with 4% PFA in PBS during 10 min. Subsequently 2% BSA and 0.1% saponin in PBS was used to block nonspecific binding. Plates were incubated with primary antibodies (rabbit‐anti‐Olig2, Merck Millipore AB9610; 1:1000 and mouse‐anti‐MBP, Biolegend SMI‐94, 1:1000) overnight at 4°C, washed with PBS, followed by incubation with alexafluor‐594 and ‐488 conjugated secondary antibodies (Life technologies; 1:1000) for 1 hr at room temperature. Cell nuclei were counterstained with Hoechst 33342 (Sigma) and wells were embedded in Fluorsave (Merck Millipore, 345789).
For each well, six adjacent fields were photographed (×10), starting at a fixed distance of the well edges. Olig2‐ and Hoechst‐positive cells were counted automatically using the analyze particles function in ImageJ v1.47. MBP area was determined using manual threshold analyses in ImageJ software v1.47. In order to reliably compare independent experiments, all results were normalized for the positive control (MCM + LPS; insert without MSCs/ 0 ng factor).
For each well, six adjacent fields were photographed (×10), starting at a fixed distance of the well edges. Olig2‐ and Hoechst‐positive cells were counted automatically using the analyze particles function in ImageJ v1.47. MBP area was determined using manual threshold analyses in ImageJ software v1.47. In order to reliably compare independent experiments, all results were normalized for the positive control (MCM + LPS; insert without MSCs/ 0 ng factor).
3
Cerebral Hypoxia-Ischemia in Mice
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Cerebral HI was induced in GFAP−/−Vim−/− and WT mice on a C57BL6−129Sv−129Ola mixed genetic background at PND5. Pups were anesthetized with isoflurane and the left common carotid artery was ligated. After surgery, mice were returned to their dams for 1 h, before being placed in a chamber with circulating humidified air (36 °C). In the chamber, mice were exposed to 10 min of air, followed by 60 min of hypoxia (10% O2 in 90% N2) followed by another 10 min of air. Following the hypoxic exposure, pups were returned to their dams until sacrifice. The brains were collected at PND14 and kept in 6% buffered formaldehyde (Histofix; Histolab) until dehydration and paraffin embedding. For the immunohistochemistry analysis, brains were sectioned at 7-μm coronal thickness on a microtome and stained against microtubule-associated protein-2 (MAP-2; clone HM-2, 1:1,000; Sigma-Aldrich catalog # M4403) or myelin basic protein (MBP; clone SMI-94, 1:1,000; BioLegend catalog # 836504).
4
Comprehensive Neuromuscular Characterization
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All human tissues in this study were acquired and processed under appropriate consent and institutional research ethics cover (REC reference: (6) . Quantitative western blotting on muscle lysates using an antibody against alpha-dystroglycan (IIH6) was performed as per a previously described protocol (7) .
Paraffin embedded samples from the brain were sectioned at 4 µm and immunohistochemistry performed on either the Ventana Benchmark Ultra or Discovery Ultra IHC Systems using the standard deparaffinisation protocol and the following antibodies and dilutions; Neurofilament Cocktail (Dako M0762) 1:500, SMI31 (BioLegend 80160) 1:5000, SMI32 (BioLegend 801701) 1:500, SMI94 (BioLegend 836504) 1:100, GFAP (DAKO Z0334) 1:2500, CD68 (Dako M0876) 1:100 and TRAPPC11 (Biorbyt ORB186301) 1:200.
Quantitative flow cytometric assay for alpha-dystroglycan was performed on cultured dermal fibroblasts from individual 1, in accordance with a procedure previously described (8) .
The trafficking of VSVG-GFP ts045 and quantification of the data was performed in cultured dermal fibroblasts from individual 1 as described by Koehler et al (9) . The retention using selective hooks (RUSH) assay was performed and quantified as described by Milev et al (10) .
Paraffin embedded samples from the brain were sectioned at 4 µm and immunohistochemistry performed on either the Ventana Benchmark Ultra or Discovery Ultra IHC Systems using the standard deparaffinisation protocol and the following antibodies and dilutions; Neurofilament Cocktail (Dako M0762) 1:500, SMI31 (BioLegend 80160) 1:5000, SMI32 (BioLegend 801701) 1:500, SMI94 (BioLegend 836504) 1:100, GFAP (DAKO Z0334) 1:2500, CD68 (Dako M0876) 1:100 and TRAPPC11 (Biorbyt ORB186301) 1:200.
Quantitative flow cytometric assay for alpha-dystroglycan was performed on cultured dermal fibroblasts from individual 1, in accordance with a procedure previously described (8) .
The trafficking of VSVG-GFP ts045 and quantification of the data was performed in cultured dermal fibroblasts from individual 1 as described by Koehler et al (9) . The retention using selective hooks (RUSH) assay was performed and quantified as described by Milev et al (10) .
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