ELISpot plates (Millipore, Ref# MSIPS4W10; Multiscreen HTS) were coated with 0.01% poly-L lysine for 1h at RT, followed by coating with 100 μg/ ml calf thymus DNA (Sigma Aldrich) or 10 μg/ ml calf thymus nucleosomes (Arotec diagnostics) overnight at 4°C. Blocked with PBS + 5% FCS for 2–3h at RT. Freshly isolated splenocytes or BM cells were resuspended in freshly prepared warm 15% RPMI 1640 media + antibiotics + 1mM L-glutamine at 20 × 106 cells/ ml. 1×106 total cells were plated on the top wells and serially double diluted (1:2). ELISpot plates were then incubated for 18h at 37°C with 5% CO2. Washed with PBS + 1% FCS to remove the cells and probed with a 1:500 dilution of goat anti-mouse IgG AP (Jackson Immunoresearch) for 3–4h at 4°C. After washing, plates were developed using the Vector Blue AP Substrate Kit III (Vector Labs). Spots were captured and counted using ImmunoSpot S6 Analyzer (Cellular Technology Limited, Shaker Heights, OH). For analysis of total IgG-secreting AFCs, ELISPot plates were coated with goat anti-mouse IgG (SouthernBiotech) at 5μg/ml and blocked with PBS + 5% BSA. Plates were washed with PBS + 1% BSA and probed with a 1:500 dilution of goat anti-mouse IgG AP (Jackson Immunoresearch).
Vector blue ap substrate kit 3
Manufactured by Vector Laboratories
Sourced in United States
The Vector Blue AP Substrate Kit III is a reagent system used for the detection and visualization of alkaline phosphatase enzyme activity in various biological applications. The kit contains the necessary components to produce a blue-colored precipitate at the site of the enzyme's activity.
Automatically generated - may contain errors
Lab products found in correlation
Nuclear fast red, by Vector Laboratories (3 mentions)
Vectastain abc ap kit, by Vector Laboratories (3 mentions)
Ab92323, by Abcam (2 mentions)
Ab172730, by Abcam (2 mentions)
H 5501, by Vector Laboratories (2 mentions)
Ab108252, by Abcam (2 mentions)
Target retrieval solution, by Agilent Technologies (2 mentions)
Calf thymus dna, by Merck Group (1 mentions)
Goat anti mouse igg ap, by Jackson ImmunoResearch (1 mentions)
Immunospot s6 analyzer, by Cellular Technology (1 mentions)
Goat anti mouse igg, by Southern Biotech (1 mentions)
Ab93678, by Abcam (1 mentions)
Anti rage antibody, by Abcam (1 mentions)
Anti cd31 antibody, by Abcam (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
5 protocols using vector blue ap substrate kit 3
1
Quantifying IgG-secreting Antibody-Forming Cells
2
Immunohistochemical Analysis of ACE2 and TMPRSS2 Expression
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Formalin-fixed paraffin-embedded tissue sections 5 μm thick were incubated in a 60° oven for 1 h and hydrated through a series steps of xylene and ethanol baths to water. Deparaffinized sections were boiled in antigen retrieval solution (100X citrate buffer, pH 6.0, Abcam, Cambridge, MA) for 10 min and cooled down for 20 min. Slides were then washed with hot tap water for 1 min and PBS twice for 3 min each time. After blocking in 5% normal goat serum diluted in PBS, the sections were incubated with anti-ACE2 antibody (ab108252, 1: 100, Abcam, Cambridge, MA), anti-TMPRSS2 antibody (ab92323, 1: 1000, Abcam) and the isotype- and concentration-matched normal IgG control (ab172730, Abcam) overnight at 4 °C. After 3 washes with PBST (PBS plus 0.1% Tween-20), the sections were incubated with the secondary biotinylated goat anti-rabbit IgG (1:2000) for 1 h at room temperature. The sections were then detected by alkaline phosphatase detection system (Vectastain ABC-AP kit, Vector laboratories, Burlingame, CA) and a blue reaction product was produced by incubating sections with alkaline substrate (Vector blue AP substrate kit III, Vector laboratories) for 20–40 min. The sections were also counterstained with nuclear fast red (Vector laboratories) and mounted with mounting medium (H-5501, Vector laboratories), and examined under a light microscope.
3
Immunohistochemical Staining for ACE2 and TMPRSS2
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Immunohistochemistry staining was performed as previous described.30 (link) Briefly, deparaffinized sections were boiled in 100 × antigen retrieval buffer (ab93678, Abcam, Cambridge, MA) for 20 minutes. After blocking in 5% normal goat serum diluted in PBS, the sections were incubated with anti-ACE2 antibody (ab108252, 1: 100; Abcam, Cambridge, MA, USA), anti-TMPRSS2 antibody (ab92323, 1:2000; Abcam), and isotype- and concentration-matched normal IgG control (ab172730, Abcam) overnight at 4°C. The sections were incubated with secondary biotinylated goat anti-rabbit IgG (1:2000) for one hour at room temperature. ACE2 staining was then detected using the alkaline phosphatase detection system (Vectastain ABC-AP kit, Vector laboratories, Burlingame, CA), and a blue reaction product was produced by incubating sections with alkaline phosphatase substrate (Vector blue AP substrate kit III; Vector Laboratories, Burlingame, CA, USA) for 20 to 30 minutes. The sections were counterstained with nuclear fast red (Vector Laboratories), mounted with mounting medium (H-5501, Vector Laboratories), and examined under a light microscope. For quantitation of ACE2 positive blood vessels, figures at magnification ×10 were taken from four consecutive fields adjacent to the optic nerve. The number of ACE2 positive vessels was quantified by two independent observers in a masked fashion.
4
Immunohistochemical Analysis of RAGE and CD31
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Deparaffinized sections were boiled in 1× target retrieval solution (Dako, Capenteria, CA) for 20 min. Endogenous peroxidase activity was quenched by treating samples with 3% hydrogen peroxide for 5 min. After blocking in 5% normal goat serum diluted in PBS (1X; 155 mM NaCl, 1.06 mM KH2PO4, 2.97 mM Na2HPO4-7H2O, pH 7.4), the sections were incubated with anti-RAGE antibody (1:125, Abcam, Cambridge, MA), anti-CD31 antibody (1:100, Abcam), or the isotype- and concentration-matched normal immunoglobulin G (IgG) control (Abcam) overnight at 4 °C. After three washes with PBST (PBS plus 0.1% Tween-20), the sections were then incubated with the secondary biotinylated goat anti-rabbit or anti-mouse IgG (1:2,000) for 1 h at room temperature. Sections were then detected with the alkaline phosphatase detection system (Vectastain ABC-AP kit, Vector Laboratories, Burlingame, CA), and a blue reaction product was produced by incubating sections with the alkaline phosphatase substrate (Vector Blue AP Substrate Kit III, Vector Laboratories). Sections were also counterstained with Nuclear Fast Red (Vector Laboratories).
5
CD8+ T Cell Immunolocalization in Ovarian Cancer
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Immunolocalization of CD8+ T cells was performed on 150 paraffin and 38 frozen sections prepared from advanced-stage serous ovarian cancer tissues using a monoclonal mouse, anti-human CD8 antibody (clone C8/144B, 1:50 dilution, DAKO, Santa Clara, CA, USA). In brief, tissue sections (paraffin-embedded) were deparaffinized, dehydrated, and antigen retrieval performed in Target Retrieval Solution (DAKO) with the pressure cooker at 120 °C for 20 minutes. Alternatively, frozen sections were fixed in methanol for 10 minutes. After blocking, sections were incubated with the primary antibody (anti-CD8) at room temperature for 90 minutes, washed two times with 1× Tris-Buffered Saline (TBS) (Boston BioProducts, Boston, MA, USA) and incubated with Polymer-AP (DAKO) for 30 minutes. CD8+ signals were visualized using a Vector Blue AP Substrate Kit III (Vector Laboratories, Burlingame, CA, USA). Digital photomicrographs of 10 fields of each slide were taken at ×20 magnification. Quantitative measurement of CD8+ cell density was determined using the Image-Pro Plus software (version 5.1; MediaCybernetics, Rockville, MD, USA).
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