Formalin-fixed, paraffin-embedded small intestine tissue sections were deparaffinized using standard laboratory protocol. Epitope retrieval solution and steamer (IHC-World, Woodstock, MD, USA) were used for antigen epitope retrieval of the tissue sections. 3% H2O2 solution was used to block the endogenous peroxidase activity for 5 minutes, followed by serum blocking (5% goat serum, 1hr). The primary antibody for α-SMA (Abcam, Cambridge, MA), TGF-β, vimentin (Santa Cruz Biotechnology, Dallas, TX) were used overnight in recommended dilutions (1:300). Species-specific biotinylated conjugated secondary antibody and streptavidin-conjugated with HRP were used to perform antigen-specific immunohistochemistry according to the manufacturer’s standard protocols. 3, 3’ Diaminobenzidine (DAB) (Sigma-Aldrich, St. Louis, and MA) was used as a chromogenic substrate. Tissue sections were counter-stained with Mayer’s hematoxylin (Sigma- Aldrich). Tissue sections were washed with 1X PBS-T (Phosphate buffered saline+ 0.05% Tween 20) between the steps. Sections were finally mounted in Aqua mount (Fisher Scientific) and observed under a 20X objective using an Olympus BX43 microscope (Olympus, America). Morphometric analysis was done using CellSens Software from Olympus America (Center Valley, PA).
Aquamount
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada, Germany
Aquamount is a water-based mounting medium used for the preparation of biological samples for microscopic examination. It is designed to provide a clear, durable, and non-toxic solution for mounting specimens on microscope slides.
Automatically generated - may contain errors
Lab products found in correlation
Axio imager, by Zeiss (6 mentions)
Lsm 880, by Zeiss (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Alexa fluor 488 antibody, by Thermo Fisher Scientific (2 mentions)
Axio imager a1 microscope, by Zeiss (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (2 mentions)
Alexa conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 donkey anti rabbit, by Thermo Fisher Scientific (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Axiovert microscope, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Epitope retrieval solution, by IHC World (1 mentions)
Vimentin, by Santa Cruz Biotechnology (1 mentions)
Mayer s hematoxylin, by Merck Group (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Tgf β, by Santa Cruz Biotechnology (1 mentions)
Cellsens software, by Olympus (1 mentions)
55 protocols using aquamount
1
Immunohistochemical Analysis of Small Intestine
2
Immunofluorescence Staining Protocol
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The cells were fixed with PBS solution containing 4% paraformaldehyde for 15 min at room temperature and then permeabilized with a solution of DMEM containing 0.05% saponin. The slides were incubated for 1 h with a primary antibody. After several washes, the plates were incubated for 1 h in the dark in the presence of an anti-mouse or anti-rabbit Alexa Fluor 488 antibody (Invitrogen). Cells were then mounted on slides using a solution of Aqua-mount (Fisher Scientific, Ottawa, Canada) and observed using a confocal microscope (LSM510META).
3
Immunocytochemistry of CND1 Protein
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Tissue sections (40 μm) were cut on a sliding microtome, placed in 0.1 M phosphate buffer, and then processed for immunocytochemistry as described previously (Westenbroek et al. 1998 (link)). Briefly, tissue sections were rinsed in 0.1 mol/L Tris buffer (TB) pH 7.4 for 15 min, in 0.1 mol/L Tris buffered saline (TBS) for 15 min, blocked using 2% avidin in TBS for 30 min., rinsed in TBS for 30 min., blocked in 2% biotin in TBS for 30 min., and finally rinsed in 0.1M TBS for 30 min. The tissue sections were then incubated in affinity‐purified anti‐CND1 (diluted 1:50) for 36 h at 4°C. All antibodies were diluted in a solution containing 0.1% Triton X‐100 and 1% NGS in 0.1M TBS. The tissue sections were rinsed in TBS for 60 min and incubated in biotinylated goat anti‐rabbit IgG diluted 1:300 for 1 h at 37°C. The tissue was then rinsed in TBS for 30 min, incubated in Streptavidin 555 diluted 1:750 for 1 hr at 37°C, then rinsed with TBS for 10 min., rinsed with TB for 20 min and mounted on charged microscope slides (Fisherbrand Superfrost/Plus), and coverslipped with AquaMount (Fisher).
4
Developmental Neurological Tissue Analysis
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Midsagittal and coronal brain sections (6 μm) at D12, D19, D40, and D60 p.b were probed with NeuN and GFAP specific antibodies, followed by labeling with Alexa 488 secondary antibody (Invitrogen) and DAPI and TUNEL for immunofluorescence, or biotinylated secondary antibody, developed with ExtrAvidin peroxidase immunostaining kit (Sigma), for immunohistochemistry. Non-specific Toluidine blue and hematoxylin staining was also performed. The slides were mounted with Aqua-mount (Fisher Scientific) and observed using an Epi-fluorescence (Olympus BX60) or bright-field microscope, and images were captured using a digital camera (DP70).
5
Immunofluorescence Staining of Cells
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Cells were fixed with PBS solution containing 4% paraformaldehyde for 15 minutes at room temperature and then permeabilized with a solution of DMEM containing 0.05% saponin. The slides were incubated for 1 hour with a primary antibody. After several washes, the plates were incubated for 1 hour in the dark in the presence of an anti-mouse or anti-rabbit Alexa Fluor 488 antibody (Invitrogen). Cells were then mounted on slides using a solution of Aqua-mount (Fisher Scientific, Ottawa, Canada) and observed using a confocal microscope (LSM510META).
6
Quantifying Micronuclei Formation and Mitotic Dynamics
7
Visualization and Quantification of Pancreatic α-Cells
8
Quantifying Fluorescence Intensity in Zebrafish
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The Tg(gcga:GFP) zebrafish larvae or immunofluorescence-stained larvae were fixed in 4% paraformaldehyde in PBS overnight and then flat mounted in Aqua-Mount (Richard-Allan Scientific) with their right side facing the coverslip. All the images were collected using a confocal microscope (Lecia Wetzlar, SP8). In addition, all the images were captured under identical criteria. Fluorescence intensity was analyzed by ImageJ software (National Institutes of Health). The total fluorescence value was analyzed based on a constant threshold. The representative rainbow images show the different intensity range in the different groups. The α-cell numbers of islets were counted manually based on the captured images. The intensity per cell was calculated using the following equation: intensity/cell= total intensity of the frame/total number of cells in the same frame.
9
Quantifying α-cell number in zebrafish larvae
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The Tg(gcga:GFP) and gcgr−/−; Tg(gcga:GFP) larvae were incubated with 20 mM glucose or a 0.3× Danieau solution from 4 to 7 dpf for three days. After harvest, larvae were fixed with 4% paraformaldehyde overnight at 4 °C, and then placed on a slide with aqua-mount (Richard-Allan Scientific, Kalamazoo, MI, USA) with the right side of larvae up to expose the islet. The α-cell number was counted according to the GFP under a Zeiss AxioImager A1 microscope (Carl Zeiss, Jena, Germany) with 40× lens.
10
Quantification of β-cell Nuclei in Larvae
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Counting of β-cells was performed as described previously (23 (link),24 (link)). In brief, after fixation in 4% paraformaldehyde overnight in 4°C, larvae were washed with 1× PBS plus 0.1% Tween-20 (PBST) and flat mounted in Aqua-Mount (Richard-Allan Scientific) with their right side facing the coverslip. The larvae were slightly flattened to facilitate counting of individual nuclei. The β-cells were counted using the nuclear mCherry signal using a Zeiss AxioImager under a 40× lens or using confocal projections taken by a Zeiss LSM710 under a 40× lens (Carl Zeiss).
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