Wild-type and R6/2 mice were sacrificed by cervical dislocation; brains were surgically removed from the skull and trimmed by removing the olfactory bulbs and spinal cord. The remaining brain were then weighed (in mg), processed and embedded in paraffin wax, and 10 μm coronal sections were cut on an RM 2245 microtome (Leica Microsystems, Srl, Milan, Italy). Five mice/group (n = 5) were used and four coronal sections spread over the anterior-posterior extent of the brain (100–200 μm inter-section distance) were scanned. For each coronal section, a total number of 10 fields at 63× magnification was randomly acquired and then analysed. Immunostaining for mutant Htt aggregates was carried out using EM48 antibody (1:100; Millipore, Darmstadt, Germany) 24 (link). Htt inclusions were defined as EM48-positive staining at the light microscope level. The average area of striatal mHtt aggregates per brain section, for each mouse brain, was quantitated by ImageJ software and reported as mHtt aggregates area (μm2) 25 (link).
Em48 antibody
Manufactured by Merck Group
Sourced in Germany, United States
The EM48 antibody is a laboratory reagent manufactured by Merck Group. It is designed for use in various immunological and biochemical applications. The EM48 antibody functions as a specific binding agent for its target analyte, enabling detection and quantification in research and analytical procedures.
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Lab products found in correlation
Rm2245 microtome, by Leica (1 mentions)
Rm2245, by Leica (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by GE Healthcare (1 mentions)
Anti erk, by Cell Signaling Technology (1 mentions)
Anti phospho erk, by Cell Signaling Technology (1 mentions)
Ecl prime, by GE Healthcare (1 mentions)
Quantity one, by Bio-Rad (1 mentions)
Anti α tubulin, by Abcam (1 mentions)
Anti darpp 32, by Cell Signaling Technology (1 mentions)
Anti bdnf, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Exoquick exosome precipitation solution, by System Biosciences (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Olympus (1 mentions)
4 protocols using em48 antibody
1
Quantifying Mutant Huntingtin Aggregates
2
Quantifying Huntington's Disease Protein Aggregates
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Wild-type and R6/2 mice were sacrificed by cervical dislocation. Brains were removed and trimmed by removing the olfactory bulbs and spinal cord. The remaining brain was weighed, processed, and embedded in paraffin wax, and 10 mm coronal sections cut on an RM 2245 microtome (Leica Microsystems). Three or four mice/group were used, and four coronal sections spread over the anterior–posterior extent of the brain (200–300 µm intersection distance) were scanned. Immunostaining for mutant HTT aggregates was carried out by using EM48 antibody (1:100) (Millipore) as previously described57 (link). The average area of striatal mHTT aggregates per section for each mouse brain was quantified with ImageJ software (developed by Wayne Rasband, National Institutes of Health, USA) and reported as aggregate area (μm2)56 (link).
3
Western Blot Analysis of Protein Expression
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40 μg of total protein lysate were resolved on SDS-PAGE and immunoblotted with specific antibodies. Anti-phospho-ERK (1:1000) and anti-ERK (1:1000) (all from cell signaling) were used for analysis of the kinase activation. For the analysis of DARPP-32, BDNF expression total lysate were immunoblotted with the anti-DARPP-32 (1:1000; Cell Signaling), anti-BDNF (1:500; Santa Cruz Biotechnology), respectively. Mutant huntingtin aggregates were detected using EM48 antibody (1:1000; Millipore). Anti-aTubulin (1:5000; Abcam, plc, Cambridge, UK) or anti-β-actin (1:3000; Sigma-Aldrich) antibodies were used for protein normalization. HRP-conjugated secondary antibodies (GE Healthcare, Buckinghamshire, UK) were used at 1:5000 dilution. Protein bands were detected by ECL Prime (GE Healthcare) and quantitated with Quantity One (Bio-Rad Laboratories) and/or ImageJ software.
4
Exosome Isolation and mHtt Aggregation Analysis
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To deplete exosomes from the mice serum, all centrifugation steps were performed at 4 °C. Exosomes were isolated by Exo-quick exosome precipitation solution (System Biosciences, Mountain View, CA), according to manufacturer's speci cations. Brie y, the serum 250μl was mixed thoroughly with 63μl of Exo-quick exosome precipitation solution and centrifuged at 1500g for 30 min. The supernatant was then removedand centrifuged at 1500g for 5 min after adding buffer. The remaining exosome pellets were then suspended in 100μl PBS. Exosome concentration was measured using BCA protein assay kitfor treatment. HD cells were treated with 200 μg/ml of young or old serum-exo and young or old serum at 2 days of differentiation and incubated for 3 days. Control groups were treated with same volume of PBS.
Analysis of mHtt aggregation in cells mHtt aggregation was quanti ed by uorescent immunocytochemistry. Cells were stained with Em48 antibody (1:400, Millipore, Billerica, MA, USA) after xing with 4% paraformaldehyde. The cells were then counterstained with DAPI (1:300, Sigma, Deisenhofen, Germany). For the uorescence staining analysis, we performed three independent experiments and over 500 cells are counted in each group. [22] Em48 (red) or DAPI (blue)-stained cells were counted using an inverted microscope (BX61, Olympus Corporation, Tokyo, Japan).
Analysis of mHtt aggregation in cells mHtt aggregation was quanti ed by uorescent immunocytochemistry. Cells were stained with Em48 antibody (1:400, Millipore, Billerica, MA, USA) after xing with 4% paraformaldehyde. The cells were then counterstained with DAPI (1:300, Sigma, Deisenhofen, Germany). For the uorescence staining analysis, we performed three independent experiments and over 500 cells are counted in each group. [22] Em48 (red) or DAPI (blue)-stained cells were counted using an inverted microscope (BX61, Olympus Corporation, Tokyo, Japan).
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