The spleen of immunized mice was separated on day 7 after the final immunization and embedded in optimal cutting temperature (OCT) compound (SAKURA). The tissues were frozen with liquid nitrogen before sectioning (7 µm) on a cryostat. After being fixed in cold acetone and blocked with 5% FBS in PBS at RT for 1 h, the sections were incubated with Biotinylated PNA (VECTOR, 1:100) overnight at 4°C. DyLight 488 Streptavidin (BioLegend, 1:100) was used as the secondary antibody at RT for 1 h followed with Alexa Fluor647-conjugated anti-mouse CD45R (BioLegend, 1:150) at RT for 1 h. After staining, the sections were scanned under a Pannoramic SCAN instrument (3DHISTECH, Hungary).
Pannoramic scan instrument
Manufactured by 3DHISTECH
Sourced in Hungary
The Pannoramic SCAN instrument is a digital slide scanner designed for high-throughput digitization of histological and cytological samples. It is capable of capturing high-resolution images of entire glass slides with a focus on speed and efficiency.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor647 conjugated anti mouse cd45r, by BioLegend (3 mentions)
Dylight 488 streptavidin, by BioLegend (3 mentions)
Biotinylated pna, by Vector Laboratories (2 mentions)
Biotinylated peanut agglutinin pna, by Vector Laboratories (1 mentions)
Cryostat, by Leica (1 mentions)
Ish kit, by Boster Bio (1 mentions)
Pannoramic viewer, by 3DHISTECH (1 mentions)
4 protocols using pannoramic scan instrument
1
Immunohistochemical Analysis of Splenic Germinal Centers
2
Immunohistochemical Analysis of Murine B Cells
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The spleens of immunized mice were separated on day 7 after the final immunization and embedded in Optimal Cutting Temperature (O.C.T) compound (SAKURA: #4583). The tissues were frozen at liquid nitrogen before sectioning (7 um) on a cryostat. After being fixed in cold acetone and blocked with 5% FBS in PBS at room temperature (RT) for 1 hour, the sections were incubated with Biotinylated PNA (VECTOR: #FL-1071-5, 1: 100) overnight at 4°C. DyLight 488 Streptavidin (BioLegend: #405218, 1: 100) was used as the secondary antibody at RT for 1 hour followed with Alexa Fluor647-conjugated anti-mouse CD45R (BioLegend: #103226,1: 150) at RT for 1 hour. After staining, the sections were scanned under a Pannoramic SCAN instrument (3DHISTECH, Hungary).
3
Immunohistochemical Analysis of Germinal Centers
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The spleens from BALB/c mice were separated one week after the final immunization and embedded in Optimal Cutting Temperature (OCT) Compound (SAKURA, USA). The tissues were frozen at -80°C before sectioning (8 μm) on a Cryostat (Leica, Germany). After being fixed in cold acetone and blocked with 1% BSA in PBS at room temperature for 1 h, the sections were incubated with Biotinylated Peanut Agglutinin (PNA, 1 : 100 dilution, VECTOR, USA) at 4°C overnight. DyLight™ 488 Streptavidin (1 : 100 dilution, BioLegend, USA) was used as the secondary antibody at room temperature for 1 h. At last, Alexa Fluor® 647-conjugated anti-mouse CD45R (1 : 150 dilution, Clone RA3-6B2, BioLegend, USA) was incubated at room temperature for 1 h. The sections were scanned under a Pannoramic SCAN instrument (3DHISTECH, Hungary). For quantification the area of GCs, spleen sections of three mice from each group were analyzed by ImageJ software.
4
In Situ Hybridization for lncRNA Detection
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In situ hybridization was performed using an ISH kit from Boster (Wuhan, China). Xylene, ethanol and protease were used to immobilize and permeate cells in clinical samples (10 μm) to allow access to biotin-labelled probes. The slides were treated with 30% H2O2 and ddH2O at a ratio of 1:10 for 5 minutes, and then the nucleic acid fragments were exposed to pepsin diluted with 3% citric acid for 20 seconds. The second fixation step was followed by an incubation with 1% paraformaldehyde/0.1 M PBS. Next, the slides were incubated with prehybridization solution at 40 °C for 2 h and then incubated with the lncRNA target probe overnight at 30 °C, followed by two washes with 2× saline sodium citrate. After blocking, biotin-labelled anti-digoxin was added and incubated for 60 minutes. Finally, the slides were stained with DAB, dehydrated with 100% ethanol and xylene, and mounted with xylene-based mounting medium. The slides were recorded with a Pannoramic SCAN instrument (3dhistech, Budapest, Hungary) and analysed using Pannoramic Viewer (3dhistech, Budapest, Hungary).
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