The passive clarity technique (PACT) (Yang et al., 2014 (link)) and immunofluorescent labeling of cleared tissue were performed as previously described (Plummer et al., 2015 (link)) except for the following modifications. Following the fixation protocol described above, 2 or 3 mm-thick coronal brain slices were embedded in 4% polyacrylamide gel (A4P0). Lipids were extracted by incubation in 8% SDS in PBS at 37°C for 6 days. During the incubation, the SDS was replaced with fresh solution every other day. For immunohistochemistry, tissue was incubated with chicken anti-GFP (1:1,000; Cat.# ab13970, Abcam) and rabbit anti-dsRed (1:500; Cat.#632496, Clontech Laboratories) primary antibodies, followed by incubation with Alexa Fluor 488 donkey anti-chicken F(ab')2 fragments (1:500; Cat.# 703-546-155, Jackson ImmunoResearch Laboratories, West Grove, PA) and Alexa Fluor 568 donkey anti-rabbit F(ab')2 fragments (1:500; Cat.# Ab175694, Abcam), for 6 days each, with buffer and antibody replaced with fresh solution after 3 days. To inhibit bacterial growth, 0.03% sodium azide was included in all antibody solutions, not 0.01% as previously published (Plummer et al., 2015 (link)).
Ab175694
Manufactured by Abcam
Ab175694 is a laboratory equipment product from Abcam. It is a core component for biological research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab13970, by Abcam (1 mentions)
Ab56307, by Abcam (1 mentions)
Elyra ps 1 superresolution microscopy, by Zeiss (1 mentions)
Prolong diamond anti fade mount, by Thermo Fisher Scientific (1 mentions)
Zen 2, by Zeiss (1 mentions)
Nucblue fixed cell readyprobes reagent, by Thermo Fisher Scientific (1 mentions)
Ab150155, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Ab15277, by Abcam (1 mentions)
Fluoview fv1000, by Olympus (1 mentions)
Ab188804, by Abcam (1 mentions)
Ab7475, by Abcam (1 mentions)
L7543, by Merck Group (1 mentions)
4 protocols using ab175694
1
Tissue Clarification and Immunolabeling Protocol
2
Immunohistochemical Staining Protocol for Cellular Structures
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4% Paraformaldehyde (PFA). Phosphate Buffered Saline (PBS). Ethanol (50%, 70%, 80%, 95%, 100%). Ethanol: Xylene (2:1, 1:1; 1:2). Xylene (100%). Xylene: Paraffin (2:1, 1:1, 1:2). Paraffin (100%). Paraffin oven. Paraffin cassettes. Rotary Microtome (Leica RM2125 RTS). Superfrost Plus Microscope Slides 15x75x1.0 mm (Fisherbrand, # 12–550 15). TRIS-buffered saline (TBS) plus 0.025% Triton X-100. 1% (Bovine serum albumin) BSA in TBS. Primary Antibodies (Cortactin (ab84208, Abcam), Tks5 (ab118575, Abcam Biotechnology), MMP-14 (ab56307, Abcam). Secondary Antibodies (Donkey F(ab’)2 Anti-Goat IgG H&L (Alexa Fluor 647) (ab150139, Abcam), Donkey F(ab’)2 Anti-Rabbit IgG H&L (Alexa fluoro-568) (ab175694, Abcam), Donkey F(ab’)2 Anti-Mouse IgG H&L 488 (ab150155, Abcam). DAPI (NucBlue Fixed Cell Ready Probes reagent, Life technologies - Catalog# R37606). Distilled water. Harris Hematoxylin solution. Lithium carbonate solution. 5% Eosin solution. Mounting medium (ProLong diamond Antifade Mount, Life technologies, #P36961). Coverslips (22mmX50mm 1.5 oz. cover class, Electron Microscopy Sciences Sciences. Catalog#72204–04. ZEISS ELYRA PS.1 super-resolution microscopy, Quorum Spinning Disk Confocal microscope. Imaging analyzing software: ZEISS ZEN 2.3 and Volocity 6.3.
3
Dystrophin Immunofluorescence Staining Protocol
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Skeletal muscles (quadriceps, gastrocnemius, and triceps) and heart were cryo-sectioned at 8 μm and fixed with 4% paraformaldehyde for 30 min. After washing with phosphate-buffered saline 3 times, 10 min each, sections were permeabilized with 0.2% Triton X-100 for 2 h and then blocked with blocking buffer containing 3% bovine serum albumin and 10% fetal bovine serum for 2 h at room temperature. Sections were then incubated with primary antibody (dystrophin, Abcam #ab15277; 1:400 dilution) overnight at 4 °C, followed by donkey anti-rabbit IgG H&L secondary antibody (Alexa Fluor 568, Abcam #ab175694) for 1 h at room temperature. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (Invitrogen; 1:1,000 dilution).
4
Quantifying Autophagy Markers in Brain Tissue
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The fixed brains were dehydratated in increasing gradients of sacharose solutions (10, 20, and 30%) and embedded in OCT Compound, sectioned coronally (25-μm thick) on cryostat, and stored in 50% glycerol in TBS at –20°C. The floating slices were permeabilized (0.1% Triton x-100, RT, 1 h), blocked (5% normal donkey serum, Abcam, ab7475; 4°C, overnight), and incubated with primary rabbit anti-LC3 antibody (1:400, Sigma-Aldrich Corp., L7543; 4°C, 48 h) following donkey anti-rabbit Alexa Fluor 568 secondary antibody (1:500, Abcam, ab175694; 4°C, overnight). Afterward, slices were mounted onto glass microscope slides with Glycerol Mounting Medium with DAPI and DABCOTM (Abcam, ab188804). Images of the hippocampus and cortex were acquired using a confocal microscope (Olympus FluoView FV 1000) with z-series followed by counting the LC3-positive puncta using Image J software (Fiji Distribution, NIH 22743772). After background subtraction, a 3D object counter plug-in with automatic thresholding was used to count objects of a volume between 6 and 250 voxels, which corresponds to puncta with a radius between 300 nm and 1 μm. The data was normalized to the volume of the slice and expressed as mean ± SD.
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