For ELISPOT assays, cells were collected from the tibia and femur bones by flushing. Red blood cell lysis was performed for 10 minutes using RBC Lysis Buffer (Biolegend) at room temperature. ELISPOT plates were prepared as previously described12 . In short, Immobilon P membrane plates (Millipore) were coated with 1 μg/ml of anti-3BNC117 overnight at 4°C. Following washing and blocking with 5% BSA in PBS, cells were seeded in dilutions of 1E5-1E6 per well, in triplicate for each mouse in RPMI 1640 (Biological Industries) supplemented with P/S (Biological Industries), 55 μM β-mercaptoethanol and 10% FCS HI (Sigma). Incubation was performed for two days at 37°C and 5% CO2. Viability at the time of harvest was commonly 60-80%. After washing, plates were incubated with anti-mouse IgG (Jackson) for 1 hour, washed again and development was performed with AEC Chromogen Kit (Sigma-Aldrich). Finally, plates were dried and incubated at 4°C until acquisition on an iSpot ELISPOT reader (AID).
Aec chromogen kit
Manufactured by Merck Group
Sourced in United States
The AEC Chromogen Kit is a laboratory reagent used in immunohistochemistry (IHC) and immunocytochemistry (ICC) applications. It provides a red-brown colored chromogenic detection system for the visualization of target antigens in tissue sections or cell samples.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon p membrane plates, by Merck Group (2 mentions)
Anti mouse igg, by Jackson ImmunoResearch (2 mentions)
Hi fcs, by Merck Group (2 mentions)
Rbc lysis buffer, by BioLegend (2 mentions)
Rpmi 1640, by Sartorius (2 mentions)
Leica cm3050, by Leica Microsystems (1 mentions)
Haematoxylin solution, by Merck Group (1 mentions)
Multiscreen hts filter plates, by Merck Group (1 mentions)
Immunospot analyzer, by Cellular Technology (1 mentions)
Ck40 32ph, by Olympus (1 mentions)
Immulon 4hbx plate, by Thermo Fisher Scientific (1 mentions)
Multiscreen ha plates, by Merck Group (1 mentions)
Antigen unmasking solution, by Vector Laboratories (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
8 protocols using aec chromogen kit
1
ELISPOT Assay for Antibody Secreting Cells
2
Immunohistochemical Analysis of UCP1
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Tissues were fixed overnight at 4°C in 4% paraformaldehyde, washed three times in PBS, infiltrated with 12.5–30% sucrose followed by optimal cutting temperature compound, and frozen in liquid nitrogen. Using a Leica CM3050 S cryostat (Leica Microsystems), 8–12-μm-thick sections were cut, fixed, and permeabilized in 10 mmol/L citric acid (pH 6.0) for 20 min at 121°C and then incubated for 20 min with 2% BSA in PBS. They were incubated for 1 h with an antibody for UCP1 (ab10983 1:2,000 dilution) followed by incubation with secondary HRP-conjugated antibodies (goat anti-rabbit IgG-HRP, 4010-05 1:200 dilution) and detected with the AEC Chromogen Kit (Sigma).
3
Immunohistochemical Analysis of Spermatogenesis
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The formalin-fixed, paraffin-embedded sections presenting normal spermatogenesis were deparaffinized in xylene (2 × 10 min) and rehydrated 2 × 5 min in 100% ethanol, 96% ethanol, and 70% ethanol. Antigen retrieval was performed as follows: incubation in 2% NaBH4 for 30 min at room temperature, incubation for 30 min in 0.1% glycine at room temperature, and incubation in 0.01% sodium citrate solution at 95 °C for the next 30 min. Afterwards, the endogenous peroxidase was blocked with 3% H2O2 (in PBS) for 30 min. After rinsing with PBS, the histological section was blocked with 10% goat serum (in PBS) for 1 h and then incubated overnight with primary antibody (diluted in 1xPBS) at 4 °C. After rinsing 2 × 5 min with PBS, the section was incubated with secondary antibody (diluted in PBS) for 1 h at room temperature. Finally, the preparation was incubated with 4 drops of AEC Chromogen Kit (Sigma-Aldrich, St. Louis, MO, USA) for 10 min; after rinsing with H2O, the haematoxylin solution (Sigma-Aldrich, St. Louis, MO, USA) was applied for 1 min. The antibody dilutions are listed in Table S4 .
4
Measuring Antibody Capture Assay
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MultiScreen HTS filter plates (Millipore) were coated with either OVA or NP1-BSA, or NP14-BSA according to manufacturer's instructions. After incubation with 1–10 × 105 cells for 4 h, captured antibodies were detected with the same detection antibodies and same dilutions as described for ELISAs and visualized with the AEC Chromogen Kit (Sigma-Aldrich). Wells were photographed with an ImmunoSpot analyzer (Cellular Technology Limited) and spots were either counted manually or using Bioreader software (BIO-SYS).
5
Histological Analysis of Skin Samples
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For histopathological observation, a portion of the skin samples from the back in each group was fixed in 10% formalin for 24 h at 4°C. After paraffin embedding, sections were cut and stained with hematoxylin and eosin (H/E) or toluidine blue (TB) for detection of infiltrated inflammatory cells or mast cells, respectively. For immunohistochemical staining, sections were incubated overnight at 4°C with the indicated primary antibodies. After sections were washed, they were incubated with horseradish peroxidase- (HRP-) conjugated anti-rabbit antibodies or HRP-conjugated anti-mouse antibodies for 1 h at room temperature. Peroxidase activity was visualized with an AEC chromogen kit (Sigma-Aldrich, St. Louis, MO, USA) and examined using a digital camera (Olympus UC30, Japan) mounted on a phase contrast microscope (Olympus CK40-32PH, Japan) using DIXI image solution 2.89 software (DIXI Optics, Daejeon, South Korea).
6
ELISPOT Assay for Antibody Secreting Cells
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For ELISPOT assays, cells were collected from the tibia and femur bones by flushing. Red blood cell lysis was performed for 10 minutes using RBC Lysis Buffer (Biolegend) at room temperature. ELISPOT plates were prepared as previously described12 . In short, Immobilon P membrane plates (Millipore) were coated with 1 μg/ml of anti-3BNC117 overnight at 4°C. Following washing and blocking with 5% BSA in PBS, cells were seeded in dilutions of 1E5-1E6 per well, in triplicate for each mouse in RPMI 1640 (Biological Industries) supplemented with P/S (Biological Industries), 55 μM β-mercaptoethanol and 10% FCS HI (Sigma). Incubation was performed for two days at 37°C and 5% CO2. Viability at the time of harvest was commonly 60-80%. After washing, plates were incubated with anti-mouse IgG (Jackson) for 1 hour, washed again and development was performed with AEC Chromogen Kit (Sigma-Aldrich). Finally, plates were dried and incubated at 4°C until acquisition on an iSpot ELISPOT reader (AID).
7
IgG1 ELISA and ELISPOT for SEA and Stag
8
Immunohistochemical Detection of NFIB
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Paraffin sections were obtained from the Department of Pathology at Hannover Medical School. Sections were deparaffinized in xylene, followed by rehydration in an ethanol gradient. Endogenous peroxidase was blocked with 3 % hydrogen peroxide in distilled water. For antigen retrieval, slides were boiled for 5 min in antigen-unmasking solution (Vector Laboratories). After washing with 0.05 % Tween-20 in Tris-buffered saline (TBST) nonspecific binding sides were blocked with blocking solution (1 % BSA with horse serum in TBST; VECTASTAIN ABC kit, Vector Laboratories) for 1 h at room temperature. Afterwards, sections were incubated overnight at 4 °C with primary antibodies against NFIB (1:300, sab1402289, Sigma-Aldrich, St. Louis, MO, USA). After repeated washings with TBST, sections were incubated with biotinylated secondary antibody Ab solution (VECTASTAIN ABC kit, Vector Laboratories) for 30 min and covered with avidin-biotin complex reagent (VECTASTAIN ABC kit, Vector Laboratories) for 1 h at room temperature. Sections were stained in aminoethylcarbazole substrate solution (AEC chromogen kit, Sigma-Aldrich, St. Louis, MO, USA) and counterstained with hematoxylin. Finally, sections were covered with mounting medium (Aquamount, VWR International GmbH, Germany). Ki-67 (1:500, Vector Laboratories, #VP-K451) was stained as previously described [2, 11] .
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