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Immpact dab solution

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ImmPACT DAB solution is a chromogenic substrate used for immunohistochemistry and immunocytochemistry applications. It produces a brown reaction product upon enzymatic conversion, which can be visualized using a light microscope.

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Lab products found in correlation

Glutaraldehyde, by Electron Microscopy Sciences (2 mentions) 1200 ex 2 transmission electron microscope, by JEOL (2 mentions) Bsa fraction 5, by Merck Group (1 mentions) Vectastain elite abc reagent, by Vector Laboratories (1 mentions) Vectamount aq aqueous mounting media, by Vector Laboratories (1 mentions) Goat anti rabbit, by Vector Laboratories (1 mentions) Triton x 100, by Merck Group (1 mentions) Ab16669, by Abcam (1 mentions) Ab93678, by Abcam (1 mentions) Ab93684, by Abcam (1 mentions) Goat anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions) Aperio cs2, by Leica camera (1 mentions) Sc 2357, by Santa Cruz Biotechnology (1 mentions) Mouse anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions) A0082, by Agilent Technologies (1 mentions) Mca341r, by Bio-Rad (1 mentions) Hematoxylin qs, by Vector Laboratories (1 mentions) G9023, by Merck Group (1 mentions) Biotinylated goat anti rabbit igg secondary antibody, by Vector Laboratories (1 mentions) P3563, by Merck Group (1 mentions)

6 protocols using immpact dab solution

1

Ultrastructural Imaging of LAMP1-APEX2 Labeled Organelles

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i3Neurons stably expressing LAMP1-APEX2 were fixed with 2% glutaraldehyde (Electron Microscopy Services) in EM buffer (0.1 N sodium cacodylate at pH 7.4 with 2 mM calcium chloride) for 30 minutes. Cells were washed 3X with EM buffer and then exposed to ImmPACT DAB solution (Vector Labs) for 10 minutes. Samples were washed with EM buffer an additional 3X and then fixed with 2% glutaraldehyde for at least an additional 48 hrs. Samples were washed with buffer and treated with 1% reduced osmium tetroxide in 0.1 N cacodylate buffer at pH 7.4 for 1 h on ice, washed and en bloc stained with 0.25–1% uranyl acetate in 0.1 N acetate buffer at pH 5.0 overnight at 4°C, dehydrated with a series of graded ethanol and finally embedded in epoxy resins. Ultrathin sections (70 nm) were stained with lead citrate and imaged with a JEOL 1200 EXII Transmission Electron Microscope.
Liao Y.C., Fernandopulle M.S., Wang G., Choi H., Hao L., Drerup C.M., Patel R., Qamar S., Nixon-Abell J., Shen Y., Meadows W., Vendruscolo M., Knowles T.P., Nelson M., Czekalska M.A., Musteikyte G., Gachechiladze M.A., Stephens C.A., Pasolli H.A., Forrest L.R., St George-Hyslop P., Lippincott-Schwartz J, & Ward M.E. (2019). RNA Granules Hitchhike on Lysosomes for Long-Distance Transport, Using Annexin A11 as a Molecular Tether. Cell, 179(1), 147-164.e20.
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2

Immunohistochemistry of Megalin and Cubilin

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Immunohistochemistry was performed on formalin fixed celloidin embedded sections. The methodology for celloidin removal and antigen retrieval steps has been described in detail (Lopez et al., 2016 (link)). Tissue sections were incubated for one hour with a blocking solution containing 5% normal goat serum/1% bovine serum albumin (BSA) fraction-V (Sigma, St. Louis, MO) and 0.5% Triton X-100 (Sigma) in PBS. Incubation with primary antibodies against megalin and cubilin was performed for 48 hours at 4°C in a humid chamber. The sections were washed with PBS (3 ×15 minutes), and then incubated for one hour with either the goat anti-rabbit or the donkey anti-goat biotinylated secondary antibodies (1:1000, Vector Labs, Burlingame, CA), and then washed with PBS (3 × 15 minutes). Next, one-hour incubation was performed with Vectastain Elite ABC reagent (Vector Labs) followed by PBS washes (15 minutes × 3). Immunoperoxidase staining was performed using Immpact DAB solution (Vector Labs). The reaction was stopped with distilled water washes (15 minutes × 4). Slides were mounted with Vectamount AQ aqueous mounting media (Vector Labs) and glass coversliped.
Hosokawa S., Hosokawa K., Ishiyama G., Ishiyama A, & Lopez I.A. (2018). Immunohistochemical localization of megalin and cubilin in the human inner ear. Brain research, 1701, 153-160.
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3

Comprehensive Histopathological Analysis of Rat Liver

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Tissue from rat livers was collected from the median and left lateral lobes, fixed with 4% paraformaldehyde, embedded in paraffin and cut to yield 2 μm sections. Afterwards the slides were deparaffinized and rehydrated with xylene and graded ethanol. Hematoxylin and Eosin (H&E), Masson's Trichrome and Picrosirius Red stainings were done according to routine protocols. For immunohistochemistry (IHC), antigen retrieval was performed with either citrate buffer (ab93678, Abcam, Cambridge, UK) or Tris-EDTA buffer (ab93684, Abcam, Cambridge, UK), depending on the antibody used. Primary and secondary antibodies were used as follows: CD3 (1:200, ab16669, Abcam, Cambridge, UK), CD19 (1:100, MAB7489, R&D Systems, Minneapolis, MN, USA), CD68 (1:100, MCA341R, Bio-Rad, Hercules, CA, USA), Von Willebrand Factor (1:200, A0082, Agilent Dako, Santa Clara, CA, USA), Fibrin (1:250, MABS2155, Sigma-Aldrich, St. Louis, MO, USA), mouse anti-rabbit IgG-HRP (1:500, sc-2357, Santa Cruz Biotechnology, Dallas, TX, USA), goat anti-mouse IgG-HRP (1:500, sc-2005, Santa Cruz Biotechnology, Dallas, TX, USA). Antibodies were visualized using ImmPACT DAB solution (Vector Laboratories, Burlingame, CA, USA) and counterstained with hematoxylin solution. The stained tissue sections were scanned with an Aperio CS2 (Leica, Wetzlar, Germany) slide scanner and analyzed with ImageJ software (Version 1.51n).
Hamberger F., Legchenko E., Chouvarine P., Mederacke Y.S., Taubert R., Meier M., Jonigk D., Hansmann G, & Mederacke I. (2022). Pulmonary Arterial Hypertension and Consecutive Right Heart Failure Lead to Liver Fibrosis. Frontiers in Cardiovascular Medicine, 9, 862330.
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4

Immunohistochemical Analysis of SOX9 in Mouse Urogenital Sinus

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Control and Sox9 cKO UGSs were sectioned and immunostained to confirm Sox9 knockout. Fixed UGS tissues were embedded in paraffin, cut into 5 μm sagittal sections, deparaffinized and rehydrated, treated with 3% hydrogen peroxide for 10 min, boiled in 10 mM sodium citrate for 20 min, and allowed to cool to room temperature to unmask epitopes. Sections were blocked for 2 h with blocking solution: 5% goat serum (Sigma-Aldrich, G9023) and 1% bovine serum albumin (EMD Millipore, 2910) in phosphate buffered saline containing 0.05% Tween-20 (PBST; Sigma-Aldrich, P3563). The Rabbit Anti-SOX9 primary antibody (Abcam ab185230) was diluted 1:250 in blocking solution, applied to the section, and incubated overnight at 4°C. Sections were washed with PBST and subsequently incubated for 1 h with biotinylated goat anti-rabbit IgG secondary antibody (Vector Labs, BA-1000) diluted 1:250 in blocking solution. Sections were washed with PBST and incubated for 30 min with peroxidase-conjugated streptavidin (Vector Labs, PK-6100). After washing with PBST, staining was achieved by incubating sections with ImmPACT DAB solution (Vector Labs, SK-4105) for 2–5 min at room temperature. Sections were counterstained for 20–30 s with Hematoxylin QS (Vector Labs, H-3404) to label nuclei.
Schneider A.J., Gawdzik J., Vezina C.M., Baker T.R, & Peterson R.E. (2019). Sox9 in mouse urogenital sinus epithelium mediates elongation of prostatic buds and expression of genes involved in epithelial cell migration. Gene expression patterns : GEP, 34, 119075.
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5

Ultrastructural Visualization of LAMP1-APEX2

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i3Neurons stably expressing LAMP1-APEX2 were fixed with 2% glutaraldehyde (Electron Microscopy Services) in EM buffer (0.1 N sodium cacodylate at pH 7.4 with 2 mM calcium chloride) for 30 minutes. Cells were washed 3X with EM buffer and then exposed to ImmPACT DAB solution (Vector Labs) for 10 minutes. Samples were washed with EM buffer an additional 3X and then fixed with 2% glutaraldehyde for at least an additional 48 hrs. Samples were washed with buffer and treated with 1% reduced osmium tetroxide in 0.1 N cacodylate buffer at pH 7.4 for 1 h on ice, washed and en bloc stained with 0.25–1% uranyl acetate in 0.1 N acetate buffer at pH 5.0 overnight at 4°C, dehydrated with a series of graded ethanol and finally embedded in epoxy resins. Ultrathin sections (70 nm) were stained with lead citrate and imaged with a JEOL 1200 EXII Transmission Electron Microscope.
Liao Y.C., Fernandopulle M.S., Wang G., Choi H., Hao L., Drerup C.M., Patel R., Qamar S., Nixon-Abell J., Shen Y., Meadows W., Vendruscolo M., Knowles T.P., Nelson M., Czekalska M.A., Musteikyte G., Gachechiladze M.A., Stephens C.A., Pasolli H.A., Forrest L.R., St George-Hyslop P., Lippincott-Schwartz J, & Ward M.E. (2019). RNA Granules Hitchhike on Lysosomes for Long-Distance Transport, Using Annexin A11 as a Molecular Tether. Cell, 179(1), 147-164.e20.
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6

Immunohistochemistry Protocols for Mouse Brain

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IHC studies were performed in paraffin-embedded coronal mouse brain (6 µm) as described in Porta et al.20 (link) Briefly, deparaffinised and rehydrated. brain sections were blocked and immunostained with the corresponding primary antibody (Table S4) overnight at 4°C. Sections were incubated for 1 h at room temperature with a biotin-conjugated secondary antibody (Table S4). Antigen-antibody reactions were visualised using VECTASTAIN AB solution and ImmPACT DAB solution (Vector Laboratories Inc., Burlingame, CA) and counterstained with haematoxylin solution. Bright-field images were acquired at 20x magnification using a Lamina Multilabel slide scanner (Perkin Elmer, Waltham, MA).
For double-label immunofluorescence, deparaffinised sections were blocked and incubated with different combinations of primary antibodies overnight at 4°C (Table S4). Secondary antibodies (Table S4) were incubated for 2 h at room temperature in a humidified chamber. To reduce endogenous autofluorescence, brain sections were immersed in a 0.3% Sudan black-70% ethanol solution for 15–20 seconds, followed by a wash in water for 10 min. Finally, sections were mounted in Vectashield-hard medium containing DAPI (Vector Laboratories Inc., Burlingame, CA). Images were obtained in a high-resolution Leica DMI6000B microscope using the Leica LAS-X software.
Porta S., Xu Y., Lehr T., Zhang B., Meymand E., Olufemi M., Stieber A., Lee E.B., Trojanowski J.Q, & Lee V.M. (2021). Distinct brain-derived TDP-43 strains from FTLD-TDP subtypes induce diverse morphological TDP-43 aggregates and spreading patterns in vitro and in vivo. Neuropathology and applied neurobiology, 47(7), 1033-1049.
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