Roti immunoblock

Cell culture medium DMEM Low Glucose, DMEM F-12, Penicillin/Streptomycin, geneticin and FBS were obtained from Biowest (Nuaillé, France). Cell culture media MCDB 131 and rhEGF were purchased from PAN Biotech (Aidenbach, Germany). Hydrocortison, ascorbic acid, DAPI, DPBS, propidium iodide and gelatin type A (300 bloom) were obtained from Merck (Darmstadt, Germany). Heparin sodium salt was purchased from Serva (Heidelberg, Germany). Sodium alginate, calcium chloride, tri-sodium citrate-dihydrate, silicon oil, Roti-Immunoblock and L-glutamine were ordered from Carl Roth (Karlsruhe, Germany). Bovine pituitary extract was obtained from Thermo Fisher/Life Technologies (Darmstadt, Germany). Resazurin sodium salt was purchased at Alfa Aesar (War Hill, GA, USA). CD31 antibody was purchased from Becton Dickinson (Heidelberg, Germany). Calcein blue AM was purchased from AAT Bioquest (Sunnyvale, CA, USA). Cyclo-Olefin Copolymer COC 5013 granulat (TOPAS Advanced Polymers GmbH, Frankfurt am Main, Germany) was a friendly gift from 3D Schilling GmbH. COC slides were purchased from Microfluidic Chipshop (Jena, Germany). Microbial transglutaminase (mTGM) was obtained from Ajinomoto (Hamburg, Germany).

For immunocytochemistry, MAC-T cells were seeded on coverslips. After overnight culture growth, coverslips were washed three times with PBS and fixed with ice-cold methanol (10 min, −20 °C). Anti-PMEL (1:100) primary antibody was incubated overnight at 4 °C. Unspecific antibody binding was blocked with Roti-Immunoblock (Carl Roth GmbH). Goat anti-rabbit IgG secondary antibody conjugated with Alexa Fluor® 488 (1:1000; # A11034, Life Technologies, Germany) was used to detect specific binding of the primary antibody. Nuclei were counterstained with 600 nM DAPI (Carl Roth GmbH). The same protocol, excluding incubation with the primary antibody, was used for the negative controls. Cells were mounted with DABCO (Carl Roth GmbH) and analyzed with the Axio Observer.Z1 inverted fluorescence microscope (Carl Zeiss Microscopy GmbH) using the Plan-Apochromat 63x/1.4 Oil DICIII objective.

Mouse cortical extracts (80 μg total protein) were electrophoretically separated on 8% (for Reelin, ADAMTS-4 and aggrecan) or 10% (for PAI-1) SDS-PAGE under reducing conditions. Proteins were transferred to nitrocellulose membranes (Bio-Rad Laboratories) and membranes were blocked for 1 h at RT with Roti®-ImmunoBlock (Carl Roth) in Tris-buffered saline. Primary antibodies [anti-Reelin, 1:1,000, MAB5364 Merck Millipore; anti-ADAMTS-4, 1:500, abcam ab185722; anti-PAI-1, 1:1,000, abcam ab222754; anti-aggrecan, 1:1,000, AB1031 Merck Millipore; anti-beta-actin clone C4, 1:4,000, MAB1501 Merck Millipore; affinity-purified anti-neuroserpin goat polyclonal antibody, 0,5 μg/ml, (Galliciotti et al., 2007 (link))] were incubated overnight at 4°C in Tris-buffered saline containing 0.05% Tween-20 and Roti®-ImmunoBlock. Secondary antibodies conjugated with IRDye® 800CW or IRDye® 680RD (1:10'000) (LI-COR Biosciences) were incubated in the same buffer for 1 h at RT. Membranes were scanned using an Odyssey® Infrared Imaging System (LI-COR Biosciences). Densitometric quantification was performed with LI-COR® Odyssey Software, version 2.0 (LI-COR Biosciences) and local background subtraction. Band intensity was normalized to beta-actin expression, and values for the wt group were arbitrarily set to 1.

Markers for secretory oviduct epithelial cells (OVGP1, PAX-8, ESR1) and epithelial markers (Cytokeratin, ß-Catenin) were evaluated by immunofluorescence (IF). Cells were grown on glass cover slips, fixed with histofix 4% (Carl Roth, Karlsruhe, Germany) overnight at 4°C, and unspecific binding sites were blocked with either 5% BSA plus 10% goat serum (Abcam, Cambridge, UK) or Roti-ImmunoBlock (1:50, Carl Roth, Karlsruhe, Germany) for 1 h at room temperature. Cells were incubated with the primary antibody overnight at 4°C. Primary antibodies and their respective dilutions (in blocking buffer) are listed in Table 1. Corresponding secondary antibodies were purchased from Thermo Fisher Scientific (Dreieich, Germany) and diluted in PBS + 1% BSA: goat anti rabbit Alexa 647 (1:200, A21245), donkey anti goat Alex 568 (1:40, A11057), and goat anti mouse Alexa 488 (1:40, A11017). Incubation time was 1 h at room temperature. Nuclei were counterstained either with TO-PRO-3 iodide (Mobitec, Berkheim, Germany) or SYBR Green I (Mobitec, Berkheim, Germany). Images were captured by the confocal laser scanning microscope LSM 800 equipped with ZEN software (Carl Zeiss, Oberkochen, Germany).

HEK-293 cell culture media and cell extracts and 80 µg mouse hippocampal extracts were electrophoretically separated on 10% SDS-PAGE under reducing conditions. Alternatively, HEK-293 cell media and cell extracts were separated on 8% non-denaturing PAGE^{19 (link)}. Proteins were transferred to nitrocellulose membranes (Bio-Rad Laboratories) and membranes were blocked for 1 h at RT with Roti-ImmunoBlock (Carl Roth) in Tris-buffered saline. Primary antibodies were incubated overnight at 4 °C in Tris-buffered saline containing 0.05% Tween-20 and Roti-ImmunoBlock. Secondary antibodies conjugated with IRDye 800CW or IRDye 680RD (LI-COR Biosciences, diluted 1:10,000) were incubated in the same buffer for 1 h at RT. Membranes were scanned using an Odyssey Infrared Imaging System (LI-COR Biosciences). Densitometric quantification was performed with LI-COR Odyssey Software, version 2.0 (LI-COR Biosciences) and local background subtraction. Band intensity was normalized to beta-actin or PDI expression. Alternatively, all proteins in the samples were quantified using Revert 700 Total Protein Stain (T.P.S) (LI-COR Biosciences).

The previously dewaxed and rehydrated liver sections (6 μm thick) were used for immunohistochemical assessment. All selected liver sections were incubated with blocking solution in phosphate buffered solution (PBS, Roti-immunoblock, Carl Roth, Karlsruhe, Germany) for 25 min at normal temperature. Subsequently, these tissues were incubated with primary monoclonal antibody for CD68 (clone ED1, abcam) and iNOS (ab3523) at dilution (1:100) for 45 min at 37°C. The liver tissues were thoroughly washed with PBS and incubated with secondary antibodies, FITC/Texas Red-conjugated goat anti-mouse IgG (1:100), for 40 min. After briefly rinsing twice with PBS, the nuclei were stained with DAPI for around a minute and then rinsed with PBS and mounted in mounting media. The immunostained tissues were analyzed by fluorescence microscopy, and their images were acquired using a Nikon DXM 1200 C camera using image analysis software AR 3.0 (Nikon, Japan). All the acquiesced images were processed using Adobe Photoshop software. The number of Kupffer cells was counted in different portions surrounding the injured area from their elongated nuclei stained with DAPI using NIS-elements software.

Formalin-fixed tissues were embedded in paraffin and sectioned at 4-μm thickness. After deparaffinization in xylene and rehydration in decreasing alcohol concentrations, sections were boiled in a citrate buffer (Dako Agilent pathology solutions, Santa Clara, CA, USA) and incubated with Roti-Immunoblock (Carl Roth, Karlsruhe, Germany). For IFM analysis, sections were incubated with primary antibodies (anti-H2A.J, Active Motif, Carlsbad, CA, USA) followed by AlexaFluor-488 secondary antibody (Invitrogen, Karlsruhe, Germany) and mounted in VECTAshield with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA). For IHC analysis, sections were incubated with anti-H2A.J antibody followed by biotin-labeled antibodies (Dako, Glostrup, Denmark) and staining was completed by incubation with 3,3′-diaminobenzidine and substrate chromogen. Finally, sections were counterstained with haematoxylin and mounted with Aqueous-Mounting-Medium (Dako, Glostrup, Denmark). For qualitative analysis, H2A.J-positive cells were visualized under the Nikon E600 epifluorescent microscope (Nikon, Düsseldorf, Germany).