6H4 methods: IHC
Immunohistochemistry on human brain sections
Sections were cut from formalin fixed, paraffin-embedded tissue blocks from cortex and cerebellum
Sections were pretreated prior to anti-PrP antibody incubation by
- hydrated autoclaving for 10 min. at 121°C,
- cooling to room temperature
- incubation in 5 min. in 96% formic acid at room temperature
- 2 hours in 4M guanidine thiocyanate at 4°C.
Alternatively, tissue sections were pretreated by
- autoclaving for 10 min in 10 mM citric acid at 121°C,
- cooling to room temperature
- incubation in 88% formic acid for 5 min. at room temperature
- incubation in 4 M Guanidine thyocyanate for 2 hours at 4°C.
mAb 6H4 was applied to the sections at a dilution of 1:500
Immunolabeling of sections was visualised using the avidin-biotin-complex method and diaminobenzidine.
Adapted from:
Kovacs GG, Head MW, Hegyi I, Bunn TJ, Flicker H, Hainfellner JA, McCardle L, Laszlo L, Jarius C, Ironside JW, Budka H (2002) Immunohistochemistry for the prion protein: comparison of different monoclonal antibodies in human prion disease subtypes. Brain Pathol 12:1-11.
Immunocytochemistry on mouse brain sections
- Brain tissue sections were treated by
- hydrated autoclaving at 121 °C for 15 min,
- immersion in 98% formic acid for 10 min
- blocking with hydrogen peroxide in methanol. The sections were then incubated in normal rabbit serum at 1:20 for 15 min.
The sections were labeled for PrP labelling, by overnight incubation with 6H4 at 1:1000 at room temperature (control sections were incubated with normal mouse serum at 1:600),
after washing biotinylated rabbit anti-mouse was added to the samples for 1 hour at room temperature
Followed by avidin-biotin comples (ABC) Elite PK-6100 and visualisation with 3’,3’diaminobenzidine (DAB). Scrapie control sections gave good staining of PrP.
From:
Baxter HC et al., (2002) Immunolocalisation of 14-3-3 isoforms in normal and scrapie-infected murine brain. Neuroscience 109: 5-14
