Tissues were fixed in 10% neutral-buffered formalin and processed for routine histological analysis. Immunohistochemistry (IHC) assays were performed using a polymer-based indirect immunoalkaline phosphatase detection system with colorimetric detection of antibody-polymer complex with Fast Red Chromogen (Biocare Medical, Concord, CA). IHC assays were performed for Ehrlichia, Anaplasma, spotted fever group Rickettsia and Leptospira species [10 (link), 11 (link)]. Tissues were also evaluated with a polyclonal rabbit sera generated against the HRTV nucleocapsid protein as previously described [1 (link)].
Fast red chromogen
Manufactured by Biocare Medical
Sourced in United States
The Fast Red Chromogen is a laboratory reagent used for the detection and visualization of target molecules in various immunohistochemical and in situ hybridization applications. It produces a bright red reaction product when catalyzed by an enzyme label, such as alkaline phosphatase. The Fast Red Chromogen is commonly used in histology and pathology laboratories to aid in the identification and analysis of specific proteins, nucleic acids, or other biomolecules within tissue samples.
Automatically generated - may contain errors
5 protocols using fast red chromogen
1
Immunohistochemical Detection of Tick-Borne Pathogens
2
Quantification of DARC/ACKR1 Gene Expression
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For in vitro DARC/ACKR1 gene expression evaluations, we used comparative quantitation methods to determine the relative mRNA levels of DARC/ACKR1 isoforms among cell lineages derived from 3 distinct ancestry groups. Specifically, after detection of the transcript with Reverse Transcription PCR, we conducted Real-time PCR using SABiosciences SYBR green protocol on Applied Biosystems 7500 cycler and SDS software version 1.4. The Ct values were transformed to dCT values using RPL13 endogenous control gene, displayed as 1/dCT to preserve directional interpretation of expression levels. We used the manufacturer’s suggested protocols for all assays and primers. Isoform specific primers were designed using the primer-BLAST program through NCBI [35 (link)]. Primer sequences were as follows:
DARC1 expression Forward-TCTGGGTATGTCCTCCAGGC
DARC1 expression Reverse-AAGGGCAGTGCAGAGTCATC
DARC2 expression Forward-TCCAATTTCCCAGCACCTCC
DARC2 expression Reverse-GGCTGGTTGGGACTACACTC
For in situ DARC/ACKR1 gene product evaluations in tissues and cells, we used immunohistochemistry to assess the DARC/ACKR1 protein expression. Formalin-fixed cultures were paraffin embedded and sections stained for DARC/ACKR1 using a goat anti-DARC antibody (Novus biologicals: NB100-2421) and detection with biotinylated anti-goat (Biocare), alkaline phosphatase streptavidin (Biogenex), and fast-red chromogen (Biocare).
DARC1 expression Forward-TCTGGGTATGTCCTCCAGGC
DARC1 expression Reverse-AAGGGCAGTGCAGAGTCATC
DARC2 expression Forward-TCCAATTTCCCAGCACCTCC
DARC2 expression Reverse-GGCTGGTTGGGACTACACTC
For in situ DARC/ACKR1 gene product evaluations in tissues and cells, we used immunohistochemistry to assess the DARC/ACKR1 protein expression. Formalin-fixed cultures were paraffin embedded and sections stained for DARC/ACKR1 using a goat anti-DARC antibody (Novus biologicals: NB100-2421) and detection with biotinylated anti-goat (Biocare), alkaline phosphatase streptavidin (Biogenex), and fast-red chromogen (Biocare).
3
Immunohistochemical Analysis of HRTV in Mice
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HRTV-inoculated Ag129 mice with signs of disease were subjected to necropsy. Spleen, liver, and kidney tissues were removed, fixed in 10% neutral-buffered formalin, and processed by using routine methods (10 (link)). Immunohistochemical analysis using rabbit polyclonal antisera against HRTV nucleocapsid (N) protein and an immuno-alkaline phosphatase detection system with Fast Red Chromogen (Biocare Medical, Concord, CA, USA) was used as described (1 (link)).
4
Immunohistochemical Detection of IEX-1 and UCP1
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Formalin-fixed paraffin-embedded tissue sections were deparaffinized and rehydrated prior to antigen unmasking by boiling in 10 mM sodium citrate and permeabilization with 0.1% TBS-Triton for 15 min. Endogenous peroxidase activity was quenched with incubation with 3% hydrogen peroxide for 15 minutes. Sections were blocked in normal goat serum and incubated with primary rabbit anti-IEX-1 (ab65152 Abcam, Cambridge MA) or anti-UCP1 (ab10983 Abcam) antibody overnight at 4 oC. Sections were then stained with biotinylated secondary antirabbit antibody for 1 hour at room temperature and color was visualized with 3,3′-diaminobenzidine (DAB) using the Vecta -stain ABC kit (Vector Laboratories). Sections for UCP1 detection were stained with polymer AP anti-rabbit secondary antibody for 30 min at room temperature (Biocare Medical, Brookline MA) and color was visualized by adding fast red chromogen (Biocare Medical). Sections were counterstained with hematoxylin prior to dehydration and coverslip placement with mounting media. The slides were scanned using a NanoZoomer and analyzed using NDP view software (Hamamatsu, Bridgewater, NJ).
5
Autopsy Evaluation for Unexplained Death
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