Immunocytochemistry

4.2.1 Immunofluorescence staining

4.2.1.1 Coating of coverslips or lumox dishes with poly-L-lysine (PLL)

Sterile PLL (4 mg/ml PBS) was used in an end concentration of 0.2 mg/ml PBS. The lumox dishes or coverslips were incubated with PLL for 1 h at RT or 30 min at 37 °C. After that the dishes or coverslips were washed 3 x with PBS for 5 min and could be used immediately for seeding of cells.

4.2.1.2 Staining of PrP^Sc

To monitor the status of the scrapie infection of the newly infected and in already infected H6-22L cells, PrP^Sc was stained in these cells and the PrP^Sc positive cells were counted afterwards.

H6-22L cells were seeded on glass coverslips to semiconfluency in a 12 well dish. They were fixed with 4 % formaldehyde solution (FA), freshly prepared from paraformaldehyde (PFA), for 20 min at RT. After pemeabilisation of the cells with 0.4 % Triton X-100 in PBS for 3 min, the cells were denatured with 6 M GdnHCl in 50 mM Tris-HCl pH 7.4 for 10 min at RT to allow the antibody (Ab) to bind PrP^Sc The unspecific binding sites for the Ab were blocked with 1 % BSA/PBS for 30 min at RT. The incubation with the 1^st Ab 6H4 1:5,000 or Kan72 1:500 diluted in 1 % BSA took place in a humid chamber for 30 min at 37 °C. Alternatively, the incubation with the 1^st Ab took place o/n at 4 °C. The coverslips were washed 3 x for 5 min with PBS before incubation with the fluorochrome labelled 2^nd Ab gαm A488 or gαr A488 diluted 1:1,000 for 30 min at 37 °C in a humid chamber. The cells were washed 1 x with PBS and then stained with 200 ng/ml Hoechst 33342 for 5 min at RT. After washing 3 x for 5 min with PBS the cells were embedded with Aqua Poly Mount.

4.2.1.3 Induction and staining of lipid bodies (LBs)

N2a or H6-22L cells were seeded on coverslips. After attachment of the cells to the coverslips (approx. 6 h later) the production of LBs was induced by addition of 200 µM oleic acid directly to the medium. After 16 h of incubation with oleic acid LBs were visible in light microscopy as big, round vesicles in the periphery of the cells. The cells were fixed with 4 % FA for 20 min at RT, permeabilised with 0.4 % Triton X-100 for 3 min at RT. Then the cells were blocked with 1 % BSA for 30 min at RT, incubated with the 1^st and 2^nd Abs and then stained for LBs with 1 µg/ml Bodipy 499/508 in PBS for 15 min at RT. The cells were washed 1 x with PBS and then stained with 200 ng/ml Hoechst 33342 for 5 min at RT. After washing 3 x for 5 min with PBS the cells were embedded with Aqua Poly Mount.

4.2.1.4 Concanavalin A (ConA) stimulation

To observe a possible endocytosis of plasma membrane bound proteins, the internalisation process was driven by ConA. ConA-A594 was diluted to 1 µg/ml in DMEM and cells were incubated for 10, 30 or 100 min at 37 °C. Unlabelled ConA was dissolved in PBS supplemented with 1 mM MgCl2 and 1 mM MnCl2 to a stock solution of 50 mg/ml. Cells were incubated for 10, 30 or 100 min at 37 °C with 100 µg/ml unlabelled ConA diluted in DMEM prior to fixation.

Methods 38

4.2.1.5 Staining of PrP^Sc in combination with trafficking organelles

N2a or H6-22L cells were seeded to semiconfluency on glass coverslips. In some cases the cells were treated with unlabelled ConA prior to fixation. The cells were fixed with 4 % FA and simultaneously permeabilised with 0.1 % digitonin for 1 h at RT. In case of H6-22L, a denaturation step with 6 M GdnHCl for 10 min followed. After blocking with 1 % BSA for 30 min at RT, the cells were incubated with the primary Ab 6H4 1:1,000 or Kan72 1:500 diluted.

Subsequently, the cells were incubated with the secondary Ab gαm-A488 or gαr-A488 each diluted 1:1,000. Afterwards the incubation with the Abs against the trafficking organelles followed: 1^st Ab gαEEA1 1:200, gαLimp2 1:50, rαERp29 1:500 or mαGM130 1:1000. The 2^nd Abs were dαg-Cy3, gαr-A546, gαm-A546, each 1:1,000 diluted. All Abs were diluted in 1 % BSA and incubated for 30 min at 37 °C in a humid chamber. After each Ab incubation the cells were washed 3 x for 5 min with PBS. The nuclei were stained with 200 ng/ml Hoechst 33342 for 5 min at RT. After washing 3 x for 5 min with PBS the cells were embedded with Aqua Poly Mount.

4.2.1.6 Assay for the detection of PrP^Sc at the plasma membrane

2 x 10⁵ N2a or H6-22L cells were grown on lumox dishes in a 24 well format, pretreated with PLL for 1 h at RT. 24 h later the cells were fixed and permeabilised with 8 % FA/0.1 % GA and 0.1 % digitonin for 1 h at RT. Cells were incubated with proteinase K (PK) in an end concentration of 20 µg/ml, diluted in PBS, for 15 min at 37 °C. The digestion was stopped with 2 mM PMSF, diluted in PBS, for 15 min at RT. In case of H6-22L, the cells were denatured with 6 M GdnHCl for 10 min. After blocking with 1 % BSA for 30 min at RT, the cells were incubated with primary and secondary Abs (6H4 1:1,000; gαm-A488 1:1,000) diluted in 1 % BSA for 30 min at 37 °C. In some cases the cells were also stained for the plasma membrane marker cholera toxin subunit B-A555 (CTB) in a dilution of 0.1 µg/ml for 10 min at RT. The nuclei were stained with 200 ng/ml Hoechst 33342 for 5 min at RT. The lumox membrane with the cells was cut out and embedded with Aqua Poly Mount on glass object slides.

4.2.1.7 Cell ELISA

N2a and H6-22L cells were seeded to confluency on a 96 well plate. The next day the cells were fixed with 4 % FA for 1 h at RT. After washing with PBS the cells were permeabilised with 0.4 % Triton X-100 for 3 min at RT. Then the cells were digested with 20 µg/ml PK (P8044) for 15 min at 37 °C. The reaction was stopped with 2 mM PMSF for 15 min at RT. The denaturation was performed with 6 M GdnHCl for 10 min at RT. After washing 3 x with TNT buffer the cells were blocked with 5 % milk powder/TNT buffer (blocking solution) for 1 h at RT. The 1^st Ab Kan72 1:1,000, 6H4 1:5,000 or 15B3 1:100 were diluted in blocking solution and incubated for 1 h at RT or o/n at 4 °C. The cells were washed 3 x in TNT buffer and then the incubation with the 2^nd Ab gαr-AP 1:2,000, gαm-AP 1:2,000 or gαm IgM-AP 1:10,000 diluted in blocking solution followed for 1h at RT. The cells were washed 3 x with TNT, followed by a washing step with 50 mM glycine-NaOH pH 9.7. Then the cells were incubated with the substrate solution until a staining in H6-22L or an unspecific staining of N2a cells appeared. The substrate solution contained BCIP and NBT. BCIP is the alkaline phosphatase (AP) substrate. After its dephosphorylation with AP, BCIP is oxidised to a dark-blue indigo-dye. NBT serves herein as the oxidant and is reduced itseld to a dark-blue indigo-dye.

4.2.1.8 Modification of the cell ELISA

To test other Abs (15B3, V5B2) for specific staining of PrP^Sc, the cell ELISA protocol was modified. The basic changes were the permeabilisation with 40 µg/ml digitonin for 3 min at RT, 0.5 % saponin, 12 min at RT or 0.2 % sarcosyl, 10 min, RT and staining with the 1^st Ab 15B3 1:100 – 1:500 diluted in growth medium without supplements or 1 %BSA prior to fixation. Also the blocking solutions were modified from 5 % milk powder/TNT, 1 % BSA, 10

% mouse serum or 10 % normal goat serum (NGS). Another variance lay in the change of washing buffers from TNT, PBS to high salt washing solution.

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4.2.2 Immunostaining for electron microscopy

4.2.2.1 Staining of PrP^C and trafficking organelles in H6-22L cells upon crosslinking with antibodies

5 x 10⁶ H6-22L cells were seeded on 6 cm lumox dishes, pretreated with PLL. The next day the medium was discarded and the cells were washed 2 x with PIPES (0.1 M PIPES pH 7.2).

The mAb 6H4 was diluted 1:500 in PIPES and the cells were incubated in vivo with the Ab for 120 min at 4 °C. The cells were washed 3 x with PIPES for 1 min. The 2^nd Ab gαm F(ab)2 Au10 nm was diluted 1:70 in PIPES and incubated for 0, 15, 60 or 180 min at 37 °C. After washing the dishes for 5min with PIPES, the cells were fixed and simultaneously permeabilised with 8

% FA/0.1 %GA first for 5 min, then in the same fixative supplemented with 0.1 % digitonin in PIPES for 55 min at RT. Cells were washed 3 x for 5 min with PIPES. PK digestion (20 µg/ml in PIPES) was performed for 15 min at 37 °C. The digestion was stopped with 2 mM PMSF in PIPES for 15 min at RT. The denaturation step was performed in an ascending and descending sequence in 1-molar steps from 1 M to 6 M GdnSCN, each step with 5 min duration. Samples were dehydrated with increasing ethanol series (30, 50, 70, 90, 95, 100

%), each for 30 min, before embedding in the methacrylate resin LR-Gold. The embedding was as follows: LR-Gold:Ethanol (1+1) o/n at 4 °C, LR-Gold for 3 h on ice, LR-Gold with 0.1 % benzil 2 x for 2h on ice. Afterwards the lumox membrane was cut into small pieces. For polymerisation the lumox pieces were put into 1.5 ml reaction tubes. The polymerisation took place by UV light first for 72 h at -30 °C (the temperature in the samples was risen to -4

°C due to exothermic reaction), then for 24 h at RT. Ultrathin sections were cut at the microtome and collected on Formvar-coated Nigrids. Some sections were stained with Limp2 and pA-Au5nm (post-embedding labelling).

4.2.2.2 Staining of PrP^Sc and trafficking organelles in H6-22L

5 x 10⁶ H6-22L cells were seeded on 6 cm lumox dishes, pretreated with PLL. The next day the medium was discarded and the cells were washed 2 x with PIPES (0.1 M PIPES pH 7.2).

The cells were fixed and simultaneously permeabilised with 8 % FA/0.1 %GA first for 5 min, then in the same fixative supplemented with 0.1 % digitonin in PIPES for 55 min at RT. Cells were washed 3 x for 5 min with PIPES. PK digestion (20 µg/ml in PIPES) was performed for 15 min at37 °C. The digestion was stopped with 2 mM PMSF in PIPES for 15 min at RT. The

denaturation step was performed in an ascending and descending sequence in 1-molar steps from 1 M to 6 M GdnSCN, each step with 5 min duration. Afterwards the cells were incubated with the mAb 6H4 1:1,000 in 1 % BSA-c/PIPES o/n at 4 °C. Then the cells were washed 3 x for 5 min with PIPES and stained with the 2^nd Ab gαm Au5nm 1:50 for 1h at 37 °C.

The cells were washed again 3 x for 5 min with PIPES, before they were dehydrated and embedded as described above. Section labelling was done with EEA1 and pA-Au10nm or Limp2 and pA-Au10nm. Then the cells were stained again with Ab 6H4 and 2^nd Ab gαm Au5nm.

4.2.2.3 Staining of PrP^Sc and trafficking organelles in H6-22L upon treatment with ConA 5 x 10⁶ H6-22L cells were seeded on 6 cm lumox dishes, pretreated with PLL. The next day the cells were pretreated with 100 µg/ml unlabelled ConA for 10, 30 or 100 min at 37 °C.

Afterwards, the cells were washed 2 x with PIPES (0.1 M PIPES pH 7.2), fixed and simultaneously permeabilised with 8 % FA/0.1 %GA first for 5 min, then in the same fixative supplemented with 0.1 % digitonin in PIPES for 55 min at RT. Cells were washed 3 x for 5 min with PIPES. PK digestion (20 µg/ml in PIPES) was performed for 15 min at37 °C. The digestion was stopped with 2 mM PMSF in PIPES for 15 min at RT. The denaturation step was performed in an ascending and descending sequence in 1-molar steps from 1 M to 6 M GdnSCN, each step with 5 min duration. After embedding, as described above, post embedding labelling was performed with Ab 6H4 and gαm-Au10 nm, EEA1 and pA-Au5nm or Limp2 and pA-Au5nm.