Mice were injected with 1.7 × 1013 GC/kg AAV2-HBKO and sacrificed 3 weeks later as described in “tissue processing and staining .” The whole spine was collected by cutting the connection to the ribs and the surrounding tissue and thereafter post-fixed overnight in 4% PFA solution. The spine was then washed 15 min in PBS and incubated for 10–14 days in 10% EDTA in PBS at 37°C. The 10% EDTA solution was changed twice a day, and the bone was examined daily until the required softness for sectioning was achieved. The spine was then washed for 15 min in PBS and post-fixed in 4% PFA for 1 h at room temperature, followed by a 15 min wash in PBS. The tissue was incubated in increasing doses of sucrose in 0.1 M PO4 going from a 10%, to 20%, and finally to 30% sucrose, with each incubation being shaken overnight at 4°C. The spine was then frozen in OCT and sectioned into 12-μm-thick sections using a Leica CM3050 S cryostat. The sections were mounted directly onto Apex Superior Adhesive slides (Leica, 3800080E-144). Stainings were performed as described in “tissue processing and staining ” except that all solutions were applied directly onto the mounted tissue and a hydrophobic pen was used to mark the edges of the tissue to keep reagents localized on tissue specimens and prevent evaporation before incubation in humidifying chambers overnight.
Apex superior adhesive slides
Manufactured by Leica
Sourced in Germany
The Apex superior adhesive slides are laboratory equipment designed to securely hold and mount specimens for microscopic examination. They feature a specialized adhesive coating that enables efficient specimen attachment and secure retention during the microscopy process.
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Lab products found in correlation
Prolong gold, by Thermo Fisher Scientific (4 mentions)
Formalin, by Avantor (3 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Mcd19 2e2b6b10 antibody, by Abcam (2 mentions)
Iview dab detection kit, by Abcam (2 mentions)
Orcaer hal100, by Hamamatsu Photonics (2 mentions)
Cm3050s cryostat, by Leica (1 mentions)
Dako autostainer plus, by Agilent Technologies (1 mentions)
Ndp view2 viewing software, by Hamamatsu Photonics (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Alizarin red, by Merck Group (1 mentions)
Hcs lipidtox deep red, by Thermo Fisher Scientific (1 mentions)
Calcein green, by Thermo Fisher Scientific (1 mentions)
Calcein blue, by Thermo Fisher Scientific (1 mentions)
7 protocols using apex superior adhesive slides
1
Spinal Tissue Preparation for Cryostat Sectioning
2
Immunohistochemical Analysis of S100A9
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4% PFA-fixed paraffin-embedded tissues were sectioned at 5 μm and mounted onto Apex Superior Adhesive Slides (Leica, Wetzlar, Germany). Standard deparaffinization was done in xylene and serial alcohol immersion. Heat-induced epitope retrieval was performed in eBioscience IHC Antigen Retrieval Solution, pH 6.0 (Thermo Scientific) by pressure cooking at 125°C for 30 s and gradual cooling to 90°C over 40 min. Slides were stained using Dako Autostainer Plus (Dako Omnis; Agilent, Santa Clara, CA, United States) slides were rinsed with buffer and blocked with H2O2 block for 10-min, Dako envision kit Protein Block (Dako Omnis). Slides were incubated with S100A9 primary antibody (1:3000 dilution, NB110–89726; Novus Biologicals) for 1 h with a posterior 30-min Dako Rabbit Labeled Polymer secondary antibody (Dako Omnis), washed (×2) for 7-min with Dako DAB + Chromogen (Dako Omnis), and counterstaining with blue Mayer’s Hematoxylin (Sigma Aldrich). Slides were imaged and scanned using a NanoZoomer 2.0-HT. Images were analyzed by NDP.view2 Viewing software (Hamamatsu Photonics, Hamamatsu, Japan) and ImageJ.
3
Perfusion-based Brain Tissue Processing
4
Multimodal Bone and Adipocyte Analysis
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Live bone staining of dissected ceratohyal bones was performed by treating with 50 µg/ml Alizarin Red (Sigma Aldrich, cat. no. A5533), Calcein Green (Thermofisher Scientific, cat. no. C481), or Calcein Blue, AM (Thermofisher Scientific, cat. no. C1429) for 5 min and repeatedly rinsing in embryo medium as described (Paul et al., 2016 (link)). We performed adipocyte labeling of dissected ceratohyal bones by incubating in a 1:200 solution of HCS LipidTOX Deep Red (Life Technologies, cat. no. H34477) for 15 min and rinsing in embryo medium as described (Minchin and Rawls, 2017 (link)). Paraffin embedding and histology were performed as described (Paul et al., 2016 (link)). We cut blocks into 5 μm sections on a Shandon Finesse Me+ microtome (cat. no. 77500102) and collected sections on Apex Superior Adhesive slides (Leica Microsystems, cat. no. 3800080). Pentachrome and Trichrome staining were performed according to manufacturer's instructions (Movat-Russell modified pentachrome stain kit, Newcomer Supply cat. no. 9150A; Gomori One-Step, Aniline Blue, trichrome stain kit, Newcomer Supply cat. no. 9176A).
5
Immunohistochemistry of Mouse Organs
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Spleens and thymuses obtained from experimental mice were immersed in 20 ml of formalin (VWR) for 24 h and transferred to PBS. Paraffin embedding was carried out using standard procedures on a Tissue-TEK VIP processor (Miles Scientific), and 4 μm sections were mounted on Apex superior adhesive slides (Leica Microsystems) and stained on a Ventana BenchMark automated IHC stainer. Immunohistochemistry sections were mounted with mounting medium, antifade reagent (Pro-Long Gold; Invitrogen) was applied, and coverslips were sealed before acquisition of images at 25 °C on a Zeiss Axiovert 200 M inverted confocal microscope with a 40 Plan Neofluor objective using IP Lab 4.0 software (Scanalytics).
6
Immunohistochemical Analysis of Murine Spleen and Liver
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Spleen and liver were obtained from experimental mice were immersed in 20 ml of formalin (VWR) for 24h and transferred to PBS. Paraffin embedding was carried out using standard procedures on a Tissue-TEK VIP processor (Miles Scientific), and 4-μm sections were mounted on Apex superior adhesive slides (Leica Microsystems) and stained on a Ventana BenchMark automated IHC stainer). mCD19 (2E2B6B10) antibody from Abcam was used and the antigen-antibody reaction was detected and visualized using the Ventana iView DAB detection kit, including secondary antibody and other necessary reagents. Immunohistochemistry sections were mounted with mounting medium, antifade reagent (Pro-Long Gold; Invitrogen) was applied, and coverslips were sealed before acquisition of fluorescent images at 25 degrees C on a Zeiss Axiovert 200M inverted confocal microscope with a 40 Plan Neofluor objective using IP Lab 4.0 software (Scanalytics). Photomicrographs were acquired using a Hamamatsu ORCAER HAL100 digital camera (400× original amplification).
7
Immunohistochemical Analysis of Murine Spleen and Liver
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