5 μm and 8 μm coronal sections were used for paraffin-embedded and frozen sections in all histological studies. The following antibodies were used at the stated dilutions: rabbit polyclonal anti-Iba1, 1:100 (Wako Pure Chemicals, Osaka Japan); rabbit polyclonal anti-GFP, 1:100 (Life Technologies, Grand Island, NY); rabbit polyclonal anti-Olig2, 1:250 (Chemicon, Temecula CA); mouse monoclonal anti-PCNA, 1:2000 (DAKO, Glostrup, Denmark); and rat monoclonal anti-CD31, 1:100 (Dianova, Germany), rabbit polyclonal cleaved caspase 3, 1:100 (Cell signaling, Danvers, MA), rat anti-BrdU, 1:100 (BIO-RAD, Hercules, CA). For IF staining, secondary antibodies conjugated to different Alexa-Fluor dyes (488 nm, 555 nm, 647 nm from Life Technologies, Grand Island, NY) at a dilution of 1:500 in PBS/2% BSA were applied. For nuclear counterstaining, DAPI was used (Sigma-Aldrich, St. Louis, MO).
Rabbit polyclonal cleaved caspase 3
Manufactured by Cell Signaling Technology
Sourced in Germany, United Kingdom
Rabbit polyclonal cleaved caspase 3 is an antibody that recognizes the cleaved form of caspase 3, a key enzyme involved in apoptosis (programmed cell death). This antibody can be used to detect and study the activation of caspase 3 in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor dye, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Rat anti brdu, by Bio-Rad (1 mentions)
Rabbit polyclonal anti gfp, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti pcna, by Agilent Technologies (1 mentions)
Image studio 2, by LI COR (1 mentions)
Protein assay dye reagent, by Bio-Rad (1 mentions)
Mouse monoclonal p53, by Santa Cruz Biotechnology (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Mouse monoclonal pcna, by Cell Signaling Technology (1 mentions)
Stereo lumar v12 microscope, by Zeiss (1 mentions)
Epcam, by BD (1 mentions)
Axiocam mrm3 ccd camera, by Zeiss (1 mentions)
Cellvue claret far red fluorescent cell linker kit, by Merck Group (1 mentions)
Hoechst 33342 solution, by Dojindo Laboratories (1 mentions)
Goat serum, by Abcam (1 mentions)
Fv1000 d confocal laser scanning microscope, by Olympus (1 mentions)
P erk1 2 rabbit polyclonal, by Cell Signaling Technology (1 mentions)
5 protocols using rabbit polyclonal cleaved caspase 3
1
Histological Assessment of Neuroinflammation
2
Western Blot Analysis of Apoptosis Markers
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Cell lysates were prepared in radioim-munoprecipitation assay buffer [20 mmol/l Tris-HCl (pH 7.5), 0.1% (w/v) sodium lauryl sulfate, 0.5% (w/v) sodium deoxycholate, 135 mmol/l NaCl, 1% (v/v) Triton X-100, 10% (v/v) glycerol, 2 mmol/l EDTA] supplemented with Complete protease inhibitor cocktail (Roche Molecular Biochemicals) and phosphatase inhibitors sodium fluoride (20 mmol/l) and sodium vanadate (0.27 mmol/l). Western blot analysis was performed as previously described (20 (link)). The total protein concentration was determined using the protein assay dye reagent from Bio-Rad Laboratories. Cell lysates in SDS-sample buffer were boiled for 5 min and equal amounts of total protein were analyzed by 10% SDS-PAGE and western blotting. The antibodies used in this study are mouse monoclonal p53 (1:250) from Santa Cruz Biotechnology; rabbit polyclonal cleaved caspase-3 (1:1,000), rabbit polyclonal cleaved PARP (1:1,000), mouse monoclonal GAPDH (1:1,000), and mouse monoclonal PCNA (1:1000) from Cell Signaling Technology. Proteins were detected using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NB, USA) and analyzed with Licor Image Studio 2.0 acquisition and analysis software.
3
Whole Mount Immunofluorescence and In Situ Hybridization
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For whole mount immunofluorescence staining fixed tissues were permeabilized with 0.5% TritonX-100 for 2h RT and washed with PBS. Unspecific staining was blocked by incubation in 5% normal donkey/goat serum, 0.3% BSA, 0.1% TritonX-100 in PBS 1h RT. Tissues were incubated overnight in +4 o C with the primary antibody rat polyclonal anti-mouse CD326 (EpCam, 1:1000, Pharmingen), rabbit polyclonal βGal (1:400, MP Biomedicals), rabbit polyclonal cleaved caspase 3 (1:400, Cell Signaling Technologies) or goat polyclonal sonic hedgehog antibody (1:100, R&D Systems) and detected with Alexa Fluor-488, Alexa Fluor-568 or Alexa Fluor-647 conjugated secondary antibodies For combined fluorescence and whole mount in situ hybridization analyses: Fluorescent imaging for fixed Fucci G1/G0 reporter whole mount mandibles was first done with a Zeiss SteREO Lumar.V12 microscope, NeoLumar S 0.8×/WD 80-mm objective, and Zeiss Axiocam MRm3 CCD camera. The samples where then subjected to whole mount in situ hybridization with digoxigenin-labeled probes specific for Shh or Wnt10b performed as described previously (Shirokova, et al., 2013; Fliniaux, et al., 2008; Wang and Shackleford, 1996) . Imaging of the hybridization signal was done with the same Zeiss Lumar microscope and Zeiss AxioCam ICc1 CCD camera and images were transposed.
4
Visualization of Apoptosis and ADSC Tracking in Lung Tissue
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We performed double immunofluorescence staining for CD31 and cleaved caspase-3 in lung sections. Briefly, samples were fixed in an optimal cutting temperature (OCT) compound and frozen in liquid nitrogen. Sections were cut into 10-μm sections, blocked with 10% goat serum (Abcam), and incubated with primary antibodies [CD31 rat monoclonal 1:200 (Abcam, Cambridge, UK) and cleaved caspase-3 rabbit polyclonal 1:200 (Cell Signaling Technology, Inc. Danvers, MA, USA)] in 1% bovine serum albumin (BSA) overnight at 4 °C, followed by secondary antibodies [Alexa Fluor 555 goat anti-rat and Alexa Fluor 488 goat anti-rabbit (Abcam) and Hoechst 33342 solution (Dojindo Laboratories, Kumamoto, Japan)] for nuclear staining for 30 min at ambient temperature. Sections were visualized under an FV-1000D confocal laser scanning microscope (Olympus, Tokyo, Japan).
To monitor the in vivo distribution of ADSCs, ADSCs were labeled with CellVue Claret Far Red Fluorescent Cell Linker Kits (Sigma-Aldrich) according to the manufacturer’s instructions. Labeled ADSCs were seeded in a six-well μ-slide (Ibidi, Munich, Germany) and visualized. Then, 4 × 106 labeled ADSCs per mouse were intravenously injected. Lung sections were observed as described previously.
To monitor the in vivo distribution of ADSCs, ADSCs were labeled with CellVue Claret Far Red Fluorescent Cell Linker Kits (Sigma-Aldrich) according to the manufacturer’s instructions. Labeled ADSCs were seeded in a six-well μ-slide (Ibidi, Munich, Germany) and visualized. Then, 4 × 106 labeled ADSCs per mouse were intravenously injected. Lung sections were observed as described previously.
5
Western Blotting Antibody Protocol
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Western blotting was performed as previously described.22 We used the antibodies according to the manufacturers' instructions. The primary antibodies were as follows: β‐actin mouse monoclonal (60008‐1‐Ig), SPON2 rabbit polyclonal (20513‐1‐AP), ERK1/2 rabbit polyclonal (16443‐1‐AP), JNK rabbit polyclonal antibody (51151‐1‐AP), p38 rabbit polyclonal (14064‐1‐AP), vimentin rabbit polyclonal (10366‐1‐AP), N‐cadherin rabbit polyclonal (22018‐1‐AP), E‐cadherin rabbit polyclonal (20874‐1‐AP; all from Proteintech Group, Inc), BAX rabbit polyclonal (#5023), BCL‐2 rabbit polyclonal (#9942), cleaved caspase‐3 rabbit polyclonal (#9664), cleaved PARP mouse polyclonal (#5625), p‐ERK1/2 rabbit polyclonal (#4370; all from Cell Signaling Technology), p‐p38 (D‐8; sc‐7973) and p‐JNK antibody (G‐7; sc‐6254; both from Santa Cruz Biotechnology, Inc).
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