Specimens were fixed in 4% paraformaldehyde overnight at four degrees, dehydrated and included in paraffin. Microtome sections were dewaxed and rehydrated using a xylene/ethanol gradient. For in situ hybridization, the RNAscope® Intro Pack 2.5 HD Reagent Kit Brown- Mm was used exactly as directed by the manufacturers. Probe used was Mm-Spry1 (Cat # 491,311), which targets sequences within the third exon. This is the only coding exon of mouse Spry1 gene and is completely removed in Spry1 knockout mice. For phospho-ERK immunohistochemistry, samples were subjected to antigen retrieval (95 °C for 20 min in Tris/EDTA buffer, pH 9) using a PTLink apparatus (DAKO). Staining was performed by an Autostainer device (DAKO), using anti-phospho-p44/42 MAPK (D13.14.4E) XP® Rabbit mAb from Cell Signaling Technologies at 1/200 dilution. Automated Hematoxylin–Eosin staining was performed using a Coverstainer device (DAKO).
Autostainer device
Manufactured by Agilent Technologies
Sourced in Denmark
The Autostainer device is an automated system designed for use in laboratories. It is used to perform staining procedures on slides or samples, ensuring consistent and reproducible results. The Autostainer can handle a variety of staining protocols and is capable of processing multiple samples simultaneously.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using autostainer device
1
Detailed Spry1 Gene Expression Analysis by In Situ Hybridization and Phospho-ERK Immunohistochemistry
2
Immunohistochemical Analysis of Fetal HCMV Brains
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Brain tissue biopsies were collected from four human fetuses electively aborted because of HCMV infection and from two controls aborted for Di George syndrome with associated cardiopathy (control case 1200496) or for premature rupture of membranes, anamnios, and chorioamnionitis (control case 1200094). Immunohistochemical brain analysis of controls and HCMV cases was performed on sections cut at 8 μm from paraffin blocks, using standard methods. Antigen was retrieved through two consecutive incubations (11 and 10 min) in a microwave oven for DCX or one 40-min incubation at 95 C in a heating bath for LIS1. Endogenous peroxidase activity and biotin were blocked by using the avidin-biotin and peroxidase blocking reagents (Vector Labs, Burlingame, CA, USA) for 10 min at room temperature. Primary antibodies were diluted 800-fold (DCX) or 250-fold (LIS1) in blocking buffer (3% BSA in PBS). Antibody specificity was ascertained by parallel staining experiments using either no primary antibody or a non-specific isotype control.
Staining for LIS1, DCX, and Mayer's haematoxylin counterstain was performed using an Autostainer device (Dako, Glostrup, Denmark), and resulting slides were scanned using a Panoramic 250 system (3D Histech, Budapest, Hungary) and analysed with the dedicated software (3D Histech).
Staining for LIS1, DCX, and Mayer's haematoxylin counterstain was performed using an Autostainer device (Dako, Glostrup, Denmark), and resulting slides were scanned using a Panoramic 250 system (3D Histech, Budapest, Hungary) and analysed with the dedicated software (3D Histech).
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