Tissue was fixed in periodate-lysine-paraformaldehyde, tissue blocks were paraffin-embedded, and sections were cut at 10μm for immunohistochemistry. Antigen retrieval for and Aβ was performed with formic acid treatment for two minutes. Sections were incubated overnight at 4°C with antibodies to phosphorylated PHF-tau (AT8; Pierce Endogen, Rockford IL; 1:2000) and Aβ (4G8; BioLegend, San Diego, CA; 1:100,000). Sections were washed three times with PBS (pH 7.4), and subsequently treated with biotinylated secondary antibody and labeled with a 3-amino-9-ethylcarbazol HRP substrate kit (Vector Laboratories). The sections were then counterstained with Gill’s Hematoxylin (Vector Laboratories H-3401) and coverslipped using Permount mounting medium. For tau pathology quantification, slides immunostained for ptau (AT8) were scanned at 20× magnification with a Leica Aperio Scanscope (Leica Biosystems, Richmond, IL) as previously described [13 (link)]. ImageScope (Leica Biosystems) was used to highlight the gray matter at the bottom third of the sulcus. Leica’s image analysis and automated counting software (Aperio positive pixel count, Version 9) was calibrated for staining intensity to detect AT8-immunoreactivity within the region of interest. Counts were normalized to the area measured and are presented as positive pixels per mm2 within the sulcal depth.
3 amino 9 ethylcarbazol hrp substrate kit
Manufactured by Vector Laboratories
The 3-amino-9-ethylcarbazol (AEC) HRP substrate kit is a laboratory reagent used for the detection and visualization of horseradish peroxidase (HRP) in immunohistochemical and immunocytochemical applications. The kit provides the necessary components to produce a colored precipitate at the site of HRP activity, enabling the localization of target proteins or antigens.
Automatically generated - may contain errors
Lab products found in correlation
Gill s hematoxylin, by Vector Laboratories (5 mentions)
Aperio scanscope, by Leica (2 mentions)
Permount medium, by Thermo Fisher Scientific (2 mentions)
Imagescope, by Leica (1 mentions)
Anti ptdp43, by Cosmo Bio (1 mentions)
Opal 7 color automation ihc kit, by PerkinElmer (1 mentions)
Anti iba1, by Fujifilm (1 mentions)
Alexa fluorophore, by Thermo Fisher Scientific (1 mentions)
Anti gfap, by Agilent Technologies (1 mentions)
Permount mounting medium, by Thermo Fisher Scientific (1 mentions)
α synuclein, by Merck Group (1 mentions)
Aβ 4g8, by BioLegend (1 mentions)
Ab5074 p, by Merck Group (1 mentions)
8 protocols using 3 amino 9 ethylcarbazol hrp substrate kit
1
Quantifying Tau Pathology in Alzheimer's Disease
2
Immunostaining of NUP62 and pTDP43 in Cortical Tissue
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Staining was performed as previously described (Cherry et al., 2016 (link)). Briefly, tissue blocks of cortical samples were taken from Broadman area 8/9 for all cases. Tissues were embedded in paraffin and cut into 20-µm-thick sections. For chromogenic staining, sections were incubated overnight at 4°C with anti-NUP62 antibody. Sections were treated with biotinylated secondary antibodies and labeled with a 3-amino-9-ethylcarbazol HRP substrate kit (Vector Laboratories). Sections were counterstained with Gill’s hematoxylin (Vector Laboratories, #H-3401). For multiplex immunofluorescent images, sections were incubated overnight at 4°C with anti-NUP62 (BD Transduction Laboratories, #610497) and or anti-pTDP43 (Cosmo Bio, #NC0877946) antibodies. Fluorescent labeling was carried out using the PerkinElmer Opal 7-Color Automation IHC kit (#NEL801001KT), per manufacturer’s instructions. Sections were counterstained with DAPI.
3
Immunohistochemical Profiling of Brain Tissue
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All brain tissue was processed identically by fixation in periodate-lysine-paraformaldehyde and stored at 4 °C. A tissue block from the dorsolateral frontal cortex was taken perpendicular to the superior frontal sulcus, embedded in paraffin and cut at 10 μm (AT8) or 20 μm (Iba1, CD68, GFAP). Antigen retrieval was performed by boiling sections in citrate buffer (pH 6.0) for 10 mins. Sections were incubated at 4 °C overnight with antibodies to anti-Iba1 (Wako, 1:500), anti-GFAP (Dako, 1:500), anti-PHF-tau (AT8) (Pierce Endogen, 1:2000), and anti-CD68 (Vector, 1:500). Sections were treated with biotinylated secondary antibodies then labeled with a 3-amino-9-ethylcarbazol HRP substrate kit (Vector Laboratories). Sections were counter stained with Gill’s Hematoxlin (Vector Laborities H-3401) and coverslipped with Permount mounting medium. For fluorescent stains, antibody visualization was performed using secondary antibodies bound to Alexa fluorophores (Invitrogen) at a dilution of 1:500. Sudan black at a dilution of 0.1 % was used to quench autofluorescence.
4
Immunohistochemical Analysis of Phospho-Tau
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For AT8 and CP13 phospho-tau histopathology, tissue blocks were embedded in paraffin for sectioning. Serial 10 μm sections were cut and mounted for immunohistochemistry, as described 56 (link)–58 (link). Briefly, mounted sections were incubated overnight at 4 °C in primary antibody AT8 (a mouse monoclonal antibody directed against phosphoserine 202 and phosphothreonine 205 of PHF-tau; Pierce Endogen, Rockford, IL; 1:2000), or CP13 (a monoclonal antibody directed against phosphoserine 202 of tau, considered to be the initial site of tau phosphorylation in neurofibrillary tangle formation; 1:200; courtesy of Peter Davies) followed by biotinylated anti mouse secondary antibody. P-tau was visualized with a 3-amino-9-ethylcarbazol HRP substrate kit (Vector Laboratories, Burlingame, CA). Sections were coverslipped with Permount medium (Thermo Scientific, Rockford, IL).
5
Immunohistochemical Analysis of Neurodegenerative Markers
6
Immunohistochemical Analysis of Tau Pathology
7
Immunohistochemical Analysis of Neurodegenerative Markers
8
Quantitative Immunohistochemistry of Neurodegenerative Markers
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Tissue was fixed in periodate-lysine-paraformaldehyde, tissue blocks were paraffin-embedded, and sections were cut at 10 µm for immunohistochemistry. Antigen retrieval for α-synuclein and β-amyloid was performed with formic acid treatment for two minutes. Sections were incubated overnight at 4 °C with antibodies to phosphorylated PHF-tau (AT8; Pierce Endogen, Rockford IL; 1:2000), ionized calcium binding adaptor molecule 1 (Iba1) (Wako, 1:500), and CD68 (Vector, 1:500). Sections were washed three times with phosphate-buffered saline (PBS; pH 7.4), and subsequently treated with biotinylated secondary antibody and labeled with a 3-amino-9-ethylcarbazol HRP substrate kit (Vector Laboratories, Burlingame, CA). The sections were then counterstained with Gill’s Hematoxylin (Vector Laboratories H-3401, Burlingame, CA) and subsequently cover slipped using Permount mounting medium.
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