Spinal cords were freshly collected and immediately frozen on dry ice after mouse perfusion with 0.1 M PBS. Tissues were processed as previously described[35] . Equal amounts of total protein homogenates were loaded on polyacrylamide gels and electroblotted onto PVDF membrane (Millipore). Membranes were immunoblotted with the following primary antibodies: rabbit anti-CXCR5 (1:1000; Abcam); mouse anti-β-actin (1:30,000; Chemicon); mouse anti-GFAP (1:10,000; Millipore); mouse anti-Chat (1:1000; Millipore); rabbit anti-Iba1 (1:1000; Wako); rabbit anti- pERK (1:1000; Cell signalling); mouse anti-ERK (1:1000; Cell signalling); rabbit anti-pAKT (1:1000; Cell signalling); rabbit anti-AKT (1:1000; Cell Signalling); mouse anti-GAPDH (1:10,000; Millipore) followed by HRP-conjugated secondary antibodies (Santa Cruz) and developed with Luminata Forte Western Chemiluminescent HRP Substrate (Millipore) on the Chemi-Doc XRS system (Bio-Rad). Densitometric analysis was performed with Progenesis PG240 v2006 software (Nonlinear Dynamics). Protein levels were normalised to the total amount of protein detected by red Ponceau (Sigma Aldrich) or to GAPDH, as previously published[35] . pERK, and pAKT levels were respectively normalized to total ERK, and total AKT.
Luminata forte western chemiluminescent hrp substrate
Manufactured by Merck Group
Sourced in United States
Luminata Forte Western Chemiluminescent HRP Substrate is a laboratory reagent designed for the detection of horseradish peroxidase (HRP) labeled proteins in Western blot analysis. It is a chemiluminescent substrate that emits light when exposed to HRP, allowing for the visualization and quantification of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc xrs system, by Bio-Rad (4 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Rabbit anti iba1, by Fujifilm (2 mentions)
Rabbit anti myod, by Proteintech (2 mentions)
Mouse anti gapdh, by Merck Group (1 mentions)
Mouse anti gfap, by Merck Group (1 mentions)
Rabbit anti akt, by Cell Signaling Technology (1 mentions)
Ponceau red, by Merck Group (1 mentions)
Rabbit anti p erk, by Cell Signaling Technology (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Mouse anti chat, by Merck Group (1 mentions)
Mouse anti erk, by Cell Signaling Technology (1 mentions)
Rabbit anti p akt, by Cell Signaling Technology (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Bio dot microfiltration apparatus, by Bio-Rad (1 mentions)
Cd206, by Abcam (1 mentions)
Rat anti cd68, by Bio-Rad (1 mentions)
Anti p ampk, by Cell Signaling Technology (1 mentions)
Rabbit anti arg1, by Abcam (1 mentions)
4 protocols using luminata forte western chemiluminescent hrp substrate
1
Western Blot Analysis of Spinal Cord
2
Quantitative Dot Blot Protein Analysis
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Proteins (3 μg) were directly loaded onto nitrocellulose Trans-Blot transfer 0.45 μm (Bio-Rad) membranes, depositing each sample on the membrane by vacuum filtration on a Bio-Dot Microfiltration Apparatus (Bio-Rad), as described previously [41 (link)]. Dot blot membranes were blocked with 3% (w/v) BSA (Sigma) and 0.1% (v/v) Tween 20 in Tris-buffered saline, pH 7.5, incubated with mouse monoclonal anti-NT (1:1000; Merck-Millipore), then with anti-mouse peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology Inc.). Blots were developed with Luminata™ Forte Western Chemiluminescent HRP Substrate (Millipore) on the ChemiDoc XRS system (Bio-Rad). Densitometry was done with Progenesis PG240 v2006 software (Nonlinear Dynamics). NT immunoreactivity was normalized to the actual amount of proteins loaded on each dot in the membrane as detected after Ponceau Red staining (Fluka). Values were expressed as mean ± SEM.
3
Western Blotting Analysis of Muscle Markers
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The mice were perfused with 0.1 M PBS, and their tissues were rapidly dissected. The TA tissues were snap-frozen using cooled isopentane and stored at −80 °C until needed. To prepare the samples for analysis, equal amounts of total protein homogenates were loaded onto a polyacrylamide gel and electroblotted onto a PVDF membrane (Millipore, Burlington, MA, USA), following the method previously described in [25 (link)]. The membranes were then immunoblotted with primary antibodies, followed by HRP-conjugated secondary antibodies from Santa Cruz Biotechnology (Dallas, TX, USA). The Luminata Forte Western Chemiluminescent HRP Substrate from Millipore (Burlington, MA, USA) was used to develop the blots, and the Chemi-Doc XRS system from Bio-Rad (Hercules, CA, USA) was used for visualization. The immunoreactivity was normalized to the total amount of protein loaded, as determined by Ponceau staining. The primary antibodies used in the experiment were mouse anti-Pax7 (1:1000; DSHB, Iowa City, IA, USA), rabbit anti-MyoD (1:1000; Proteintech, Rosemont, IL, USA), mouse anti-MyoG (1:130; DSHB, Iowa City, IA, USA), and CD206 (1:500; Abcam, Cambridge, UK).
4
Western Blot Analysis of Muscle Proteins
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Mice were sacrificed according to institutional ethical procedures by decapitation, and the GCM was frozen on cooled isopentane and stored at −80 °C until use. Equal amounts of total protein homogenates were loaded on a polyacrylamide gel and electroblotted onto a PVDF membrane (Millipore) as previously described [25 (link)]. Membranes were immunoblotted with the primary antibodies, followed by HRP-conjugated secondary antibodies (Santa Cruz) and developed by Luminata Forte Western Chemiluminescent HRP Substrate (Millipore) on the Chemi-Doc XRS system (Bio-Rad). Immunoreactivity was normalised to the total amount of protein loaded (Ponceau). The following antibodies were used: goat anti-Agrin (1:200; R&D Systems), rabbit anti-Rapsyn (1:1000; Abcam), mouse anti-Pax7 (1:1000; DSHB), rabbit anti-MyoD (1:1000; Proteintech), mouse anti-MyoG (1:130; DSHB), mouse anti-Sirtuin1 (1:750; Sigma Aldrich), rabbit anti-hSOD1 (1:1000; StressMarq Biosciences), rabbit anti-ARG1 (1:500; Abcam), anti-AMPK (1:1000; Cell Signaling), and anti-pAMPK (1:1000; Cell Signaling).
Immunoblot of human muscle biopsies was performed using the same protocol described above using the following primary antibody: rat anti-CD68 (1:500, Bio-Rad), rabbit anti-iNOS (1:2000, Abcam), rat anti-CD206 (1:500, Bio-Rad), and rabbit anti-Iba1 (1:500; Wako).
Immunoblot of human muscle biopsies was performed using the same protocol described above using the following primary antibody: rat anti-CD68 (1:500, Bio-Rad), rabbit anti-iNOS (1:2000, Abcam), rat anti-CD206 (1:500, Bio-Rad), and rabbit anti-Iba1 (1:500; Wako).
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