Sections were stained for e-cadherin (BD Biosciences Cat# 610181) and vimentin (BD Biosciences Cat# 550513), at a concentration of 1:200 as per method described (28 (link)), using HRP tagged secondary antibodies. Briefly, sections were deparaffinized in xylene for 10 mins followed by rehydration in alcohol gradients. Endogenous peroxidase was blocked by treatment with 3% H2O2 (Merck 17544) in methanol for 30 mins, following rehydration. Antigen retrieval was performed in 1 mM sodium citrate buffer at 100°C for 15 mins followed by cooling at RT for 15 mins. Blocking of non-specific sites was carried out by incubating the slides for 30 mins in 8% BSA-PBS. Sections were incubated overnight with primary antibodies diluted in 1% BSA followed by washing with PBS-Tween 20 and incubation with HRP tagged anti-mouse secondary antibodies. Chromogenic color was developed using AEC Substrate (Vector Laboratory SK4200) as per manufacturer’s protocol. The sections were counterstained with Hematoxylin (Merck-HX68597049) for 40 secs and mounted with Vectamount (Vector Laboratories H5501). Image acquisition was done on Carl Zeiss Plan Achromat bright field microscope, Axiocam 1058 color camera with objectives 4x, 10x and 20x.
Aec substrate
Manufactured by Vector Laboratories
Sourced in United States
AEC substrate is a chromogenic substrate used in immunohistochemistry and enzyme-linked immunosorbent assays (ELISA) to produce a red-brown colored precipitate. It serves as a detection system for visualizing the presence and localization of specific target antigens in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Maips4510, by Merck Group (2 mentions)
Hematoxylin, by Merck Group (1 mentions)
E cadherin, by BD (1 mentions)
Vimentin, by BD (1 mentions)
Glycerol gelatin aqueous slide mounting medium, by Merck Group (1 mentions)
Leica dm4000b microscope, by Leica camera (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Hematoxylin, by Vector Laboratories (1 mentions)
20 objective, by Hamamatsu Photonics (1 mentions)
Nanozoomer slide scanner, by Hamamatsu Photonics (1 mentions)
A8081, by Merck Group (1 mentions)
M7020, by Agilent Technologies (1 mentions)
Af338, by R&D Systems (1 mentions)
Pvdf membrane plates, by Merck Group (1 mentions)
Protein block, by Agilent Technologies (1 mentions)
Rabbit anti human anti mouse mcl 1 antibody y37, by Abcam (1 mentions)
Biotinylated swine anti rabbit igg, by Agilent Technologies (1 mentions)
Anti rabbit secondary antibody, by Vector Laboratories (1 mentions)
Hematoxylin, by Carl Roth (1 mentions)
Ab37415, by Abcam (1 mentions)
11 protocols using aec substrate
1
Immunohistochemical Analysis of E-Cadherin and Vimentin
2
Macrophage Quantification in Aortic Tissue
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Aortic root sections were fixed for 15 minutes in 10% methanol-free formalin and washed three times with PBS. Sections were then blocked for 30 minutes to protect against endogenous tissue peroxidase activity (Signet Covance, Prineceton, NJ), washed twice with PBS, and then blocked with Ultra V protein block (Lab Vision, Fremont, CA) for 7 minutes. The sections were next incubated in Ultra V protein block solution (1:4 in PBS) containing monoclonal antibody to macrophages-2 (MoMa-2, 1:600; Acris, Hiddenhausen, Germany) for 1 hour at RT, and then overnight at 4°C.
The sections were next washed twice with PBS, incubated for 3 hours with an HRP-coupled polyclonal rabbit anti-rat secondary antibody (1:100, Dako Denmark A/S, Glostrup, Denmark), and washed three times with PBS. AEC substrate (Vector Laboratories, Burlingame, CA) was added until reaction development and then washed away 3 times with H2O. Sections were counterstained with hematoxylin for 5 min, washed in 0.1% NaHCO3, and mounted with glycerol-gelatin aqueous slide mounting medium (Sigma-Aldrich, St. Louis, MO). Images were taken with a Leica DM 4000B microscope equipped with a Leica DFC 500 camera. The intensity and extent of MoMa-2 staining of three consecutive sections per mouse was quantified using ImageJ software.
The sections were next washed twice with PBS, incubated for 3 hours with an HRP-coupled polyclonal rabbit anti-rat secondary antibody (1:100, Dako Denmark A/S, Glostrup, Denmark), and washed three times with PBS. AEC substrate (Vector Laboratories, Burlingame, CA) was added until reaction development and then washed away 3 times with H2O. Sections were counterstained with hematoxylin for 5 min, washed in 0.1% NaHCO3, and mounted with glycerol-gelatin aqueous slide mounting medium (Sigma-Aldrich, St. Louis, MO). Images were taken with a Leica DM 4000B microscope equipped with a Leica DFC 500 camera. The intensity and extent of MoMa-2 staining of three consecutive sections per mouse was quantified using ImageJ software.
3
Immunostaining of Recombinant Baculoviruses
4
Quantification of Rotavirus-Specific Antibody-Secreting Cells
5
Quantification of Rotavirus-Specific Antibody-Secreting Cells
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Multiscreen 96-well plates (MAIPS4510, Millipore) were coated with purified rotavirus double-layer particle (gift from H. Greenberg) overnight at 4°C, washed, and blocked with RPMI medium containing 10% FCS for 1 h at 37°C. Serial 10-fold dilutions of spleen, PP, or SI LP cells were added to the plates, and incubated overnight at 37°C. After six washes, peroxidase-conjugated anti-mouse IgA or IgG were added for 1 h at 37°C. Plates were washed six times and developed with AEC substrate (Vector Laboratories). To quantity non-specific ASCs, plates were coated with anti-mouse IgA, IgG, IgM polyclonal antibodies before using the same protocol as above.
6
Immunohistochemical Tn Antigen Detection
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The ethanol-fixed human tissue sections, collected and stored before the French law 88–138 of the 20th of December 1988 on tissue resection for scientific investigation, were obtained from the Nantes University Hospital Center for Biological Resources1 (approval DC-2011-1399).
Immunohistochemistry was performed as described elsewhere (Lopes et al., 2018 ). Briefly, the slides were deparaffinized and blocked for endogenous peroxidase activity and non-specific protein binding and incubated with the IgM mouse monoclonal antibodies against Tn NaM217-2A9 that was raised against human Tn erythrocytes (Duk et al., 2001 ) overnight at 4°C. The slides were then successively incubated with HRP-conjugated anti-mouse IgG (H + L) (Uptima; Interchim, Montluçon, France) and AEC substrate (Vector Laboratories, Burlingame, CA, United States) with three PBS washes in-between and conterstained with hematoxylin (Vector Laboratories) before mounting and imaging with a Nanozoomer slide-scanner using a ×20 objective (Hamamatsu Photonics, Massy, France).
Immunohistochemistry was performed as described elsewhere (Lopes et al., 2018 ). Briefly, the slides were deparaffinized and blocked for endogenous peroxidase activity and non-specific protein binding and incubated with the IgM mouse monoclonal antibodies against Tn NaM217-2A9 that was raised against human Tn erythrocytes (Duk et al., 2001 ) overnight at 4°C. The slides were then successively incubated with HRP-conjugated anti-mouse IgG (H + L) (Uptima; Interchim, Montluçon, France) and AEC substrate (Vector Laboratories, Burlingame, CA, United States) with three PBS washes in-between and conterstained with hematoxylin (Vector Laboratories) before mounting and imaging with a Nanozoomer slide-scanner using a ×20 objective (Hamamatsu Photonics, Massy, France).
7
Immunohistochemical Profiling of Activin Signaling
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Formalin-fixed 5 μm paraffin sections were deparaffinized, and antigen retrieval was performed with 4 M hydrochloric acid (follistatin, β2-microglobulin) or proteinase K (vimentin). The tissue was permeabilized with Triton X-100 and endogenous peroxidases blocked with 0.3% H2O2 in 100% methanol. After blocking with 10% dry milk, the slides were incubated overnight with primary antibodies in 2.5% BSA at 4 °C: goat anti-human/mouse/rat polyclonal activin A (AF338, R&D, Germany), mouse anti-human monoclonal follistatin (MAB669, R&D, Germany), goat anti-human polyclonal ACVR2A (A8081) and ACVR1B (A2455) (both Sigma-Aldrich, Germany), mouse anti-human monoclonal β2-microglobulin (ab54810, Abcam, UK), and mouse anti-human monoclonal vimentin (M7020, Dako, USA). Slides were incubated 30 min with secondary antibodies (Histofine, Medac), and color development was performed with AEC substrate (Vector Laboratories, USA). For snap-frozen tissues, 5 μm acetone-fixed sections were used with the same procedure.
8
ELISpot Assay for HA-Specific ASCs
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HA-specific antibody-secreting cells (ASCs) were detected using an ELISpot protocol as previously described with modifications.19 (link) Briefly, cultures that were either stimulated with influenza antigens for 7 days or left unstimulated were resuspended and enumerated, then plated on HA-coated (see Table S5 ) and blocked 96-well PVDF membrane plates (Millipore). Each sample was plated in duplicate and total live-cell counts ranged from 3.6 x 104 to 1.08 x 105 cells per well. Cells were incubated on these membranes, undisturbed for 5h at 37°C. Plates were then washed and treated with horseradish peroxidase-conjugated anti-IgG/IgA/IgM secondary antibody (Abcam). After incubation overnight at 4°C, plates were washed and developed with AEC substrate (Vector), washed 20 times with water, dried, and spots were enumerated. The frequency of ASCs out of total B cells was determined from B cell flow cytometry data analysis and the direct cell enumeration counts.
9
Mcl-1 Immunohistochemistry Protocol
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Joints sections were cut and subjected to antigen retrieval in decloaker chamber in Diva Decloaker (Histolab) following deparaffination. Sections were blocked with Protein Block (Dako) for 30 min before incubation with Rabbit anti-human/anti-mouse Mcl-1 antibody (Y37, abcam). Peroxidase activity was blocked with 3% H2O2. Slides were incubated with biotinylated swine anti-rabbit IgG (Dako) and developed using ABC detection system with AEC substrate (Vectastain). Sections were counterstained with hematoxylin and mounted.
10
Immunohistochemistry of Proliferation and Apoptosis
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Acetone-fixed cryosection slides were prepared. Slides were incubated with rabbit polyclonal anti-Ki-67 (1:50 dilution, ab15580, Abcam, Cambridge, UK), cleaved caspase-3 (1:400 dilution, 9661, Cell Signaling, Danvers, MA, USA) antibodies diluted with DAKO diluent (Agilent Technologies, Santa Clara, CA, USA) for 60 min at RT, and washed three times with PBS-Tween-20 (0.05%) (Sigma-Aldrich, St. Louis, MO, USA). Next, secondary antibody (anti-rabbit, Vector, Burlingame, CA, USA) diluted in goat serum (Vector, Burlingame, CA, USA) and DPBS was applied to the slides and incubated for 30 min. After washing steps as described above, an avidin-biotin-complex (Vector, Burlingame, CA, USA) was added to the slides for 30 min incubation. Then, the AEC substrate (Vector, Burlingame, CA, USA) was applied to the slides and incubated for 3 min (anti-Ki-67) or 8 min (cleaved caspase-3). Finally, slides were counterstained with hematoxylin (Carl Roth GmbH, Karlsruhe, Germany) for 7 min. Ki-67 and cleaved caspase-3 positive cells were counted in ten high-power fields per slide. Rabbit IgGs (ab37415, Abcam, Cambridge, UK) served as a negative control.
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