Transverse serial cross sections (7 μm) were obtained using a cryostat maintained at −20°C. Sections were then blocked (1× PBS/5% normal serum/0.3% Triton X-100), and laminin primary antibody (Thermo Fisher Scientific, MA1-06100, 1:100) was diluted in dilution buffer (1× PBS/1% BSA/0.3% Triton X-100) and incubated overnight at 4°C. Three washes with PBS were performed before applying secondary antibody (Abcam, ab175476, 1:1,000) at room temperature for 1 h, and ProLong diamond antifade mountant was used (Thermo Fisher Scientific, 36961). Stained slides were digitalized with the Zeiss Apotome.2 microscope with a ×20 objective (Hamamatsu), and myofiber CSA was semiautomatically quantified using SMASH application on MATLAB software (v. R2021b) (17 (link)). In addition, we investigated the lipid content through oil red O staining. Only lipids outside muscle fibers were quantified (MATLAB software v. R2021b) to exclude myocellular triglyceride content and then reflect IMAT deposits. Finally, collagen area was investigated through modified Masson’s trichrome staining (18 ) and quantified with MATLAB software (v. R2021b).
Ab175476
Manufactured by Abcam
Ab175476 is a laboratory equipment product available from Abcam. It is a core piece of apparatus used for scientific experimentation and research. The detailed specifications and intended use of this product are not available in a concise, unbiased, and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
Apotome 2 microscope, by Hamamatsu Photonics (1 mentions)
Ab6326, by Abcam (1 mentions)
Vectashield h 1000, by Vector Laboratories (1 mentions)
C6891, by Merck Group (1 mentions)
A11001, by Thermo Fisher Scientific (1 mentions)
Ab6160, by Abcam (1 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
Ab3201, by Merck Group (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Ab150077, by Abcam (1 mentions)
4 protocols using ab175476
1
Histological Analysis of Skeletal Muscle
2
DNA Combing Assay for Replication Dynamics
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DNA combing was performed as previously described (Moudry et al, 2022 (link)). In brief, cells were labeled with 25 μM CldU (I7125; Sigma-Aldrich) and 250 μM IdU (C6891; Sigma-Aldrich) for 20 min. DNA was then extracted using a FiberPrep kit (EXT-001; Genomic Vision) following the manufacturer’s instructions. Extracted DNA was combed on vinylsilane coated CombiCoverslips (COV-002-RUO; Genomic Vision), denatured, dehydrated, air-dried and blocked. Coverslips were incubated with primary antibodies, mouse anti-BrdU (1:10, BD347580; BD Biosciences), and rat anti-BrdU (1:50, ab6326; Abcam) antibodies. After four washes with PBS, cover glasses were incubated with secondary antibodies goat anti-mouse Alexa Fluor 488 (1:100, A11001; Invitrogen) and goat anti-rat Alexa Fluor 568 (1:100, ab175476; Abcam) antibodies. After four washes with PBS, cover glasses were air-dried and mounted using Vectashield (H-1000; Vector Laboratories). Images of DNA fibers were acquired using CellObserver spinning disc confocal microscopic system (Zeiss), and length of labeled DNA was analyzed using ImageJ software.
3
Embryonic Explant Immunostaining
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Embryonic explant cultures were fixed and labelled [12 (link)] with primary antibodies (1:1000 diluted in blocking buffer made up by 1% non-fat dry milk in calcium and magnesium free PBS) to tyrosinated tubulin (rat monoclonal, ab6160, Abcam) and de-tyrosinated tubulin (rabbit polyclonal, AB3201, Millipore), and with the secondary antibodies goat anti-rat Alexa Fluor 568 (1:1000, ab175476, Abcam) and goat anti-rabbit Alexa Fluor 488 (1:1000, A-11008, Life Technologies), respectively.
4
Immunofluorescent Analysis of Striatal and Midbrain Tissue
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Mice were transcardially perfused with 4% paraformaldehyde in 0.1 M PBS (pH 7.4) 5 to 6 weeks after viral injections. Coronal sections (40 μm) from the striatum and midbrain were rinsed in PBS and incubated 30 minutes in retrieval buffer (10 mM trisodium citrate in water, pH 6.0) at 80°C. After washing, sections were blocked for 30 minutes (PBS with 5% goat serum, 1% BSA, 0.3% Triton X-100 in PBS) and incubated overnight (4°C) with primary antibodies against TH (Thermo Fisher Scientific, OPA1-04050 1:1000) and HA (Roche, 3F10 1:200). Sections were rinsed 3 times in washing buffer (0.25% BSA and 0.1% Triton X-100 in PBS) and incubated 60 minutes with secondary Alexa Fluor 488 and Alexa Fluor 568 antibodies (Abcam, ab150077 and ab175476, 1:400 in washing buffer). Sections from mice injected with AAV8-hSYN-DIO-mCherry were stained only for TH. Sections were mounted on glass coverslips (Mentzel-Gläzer, 24 × 60 mm) using ProLong Gold Antifade Mountant mounting media with DAPI (Life Technologies, P36931). Images were acquired using a Slide Scanner Axio Scan.Z1 (Zeiss) with 488 nm, 561 nm, and DAPI channel settings.
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