Métodos Clásicos

A ntigenic profiles of prim ary cultures of childhood b rain tum ours

Specific antibodies w ere used as m arkers for distin g u ish in g different cell ty p es using im m unocytochem ical techniques. Early passage cell lines w ere used to screen the antigenic expression by cells derived from childhood brain tum ours. Additionally, the antigenic profiles of some cultures grow n on different attachm ent factors were also screened.

These studies concentrated on interm ediate filam ents, including GFAP, vim entin, neurofilam ent, cytokeratin and fibronectin. Synaptophysin as an additional m arker w as used to

screen the m edulloblastom a cultures. A small num ber of cultures w ere screened for desmin.

The species of origin, w orking dilution and supplier are sum m arised in Table 6.

Table 6: Type and working dilution of antibodies

A n tib o d y C lass

A n tib o d y Source W o rkin g

D ilu tio n

Supplier

Prim ary GFAP m ouse (mono) 1:10 A m ersham R P N 1106

NF (160kD) mouse (mono) 1:10 A m ersham RPN 1104

VIM mouse (mono) 1:10 A m ersham RPN 1102

CYT mouse (mono) 1:10 A m ersham RPN 1100

DES mouse (mono) 1:10 A m ersham RPN 1101

FN rabbit (poly) 1:200 Dako A245

SYN rabbit (poly) 1:25 Dako AOlO

Secondary Anti-mouse Ig sheep (mono) 1:50 Am ersham RPN 1001

Anti-rabbit Ig donkey (poly) 1:50 Am ersham RPN 1004

Staining Fluorescein 1:50 A m ersham RPN 1232

Preparation of early passage cultures for im m unocytochem istry

U nder aseptic conditions, cells w ere prep ared for im m unocytochem ical staining, either on 13mm diam eter coverslips (glass) or on m ulti-cham ber slides (Nunc). Coverslips w ere plated

w ith 5 X 10'^ cells in 0.5ml m edium and the 8-slide chambers were seeded w ith 1 x 10^ cells in a

total volum e of 4ml. The cells were incubated at 37°C for 2-4 days, until 60-85% confluency w as reached. This w as im portant because, firstly, the cells w ould have reached a stage w hereby the m orphology seen w ould be that of cells during the exponential phase of grow th, secondly, the cells w ould not be too confluent to interfere w ith interpretation of the results and, thirdly, piling u p of cells w ould have been kept to a m inim um . In addition, the passage level at w hich these cultures w as screened was not beyond level five in order to be as close to the original tum our specim en as was possible.

Preparation of ECM passaged cultures for im m unocytochem istry

The same procedure was carried out as that for cultures grow n on different attachm ent factors.

H ow ever, since earlier attem pts showed that coating the 8-slide chambers w ith the appropriate

attachm ent factor seemed to interfere w ith the final results w hen view ed u n d er the microscope, it w as decided to initially grow the cultures on the relevant attachm ent factor an d then on reaching exponential grow th to seed the cells and then plate them onto the m ulti-cham ber slides w ithout the attachm ent factor. The cultures were then treated as described above.

The Biotin-Streptavidin system

The biotin-streptavidin technique w as em ployed in detecting and screening the antibody- antigen im m unocom plexes (Am ersham ). Based on the bio tin -av id in im m unoperoxidase m ethod (Amersham), this technique has been enhanced, com bining the specificity of highly purified antibody probes w ith high affinity binding of the small w ater soluble vitam in biotin to the bacterial protein streptavidin. Species-specific secondary antibodies against the prim ary im m unoglobulin w ere covalently linked to biotin. A tertiary, non-im m unological reagent com posed of biotin -strep tav id in com plex covalently b o u n d to a fluorochrom e such as fluorescein isothiocyanate (FITC) was used. A m uch im proved signal, identifying the prim ary antibody-antigen interaction, was therefore generated.

Immunofluorescence

Cell m onolayers w ere w ashed w ith HBSS or PBS (3x) and then fixed w ith 1% H C l in 70% ethanol for 10 m inutes. Following neutralisation w ith a further three w ashes w ith HBSS, the p rim ary antibody w as applied for 1-2 hours at room tem perature. Slides w ere kept in a hum idified environm ent to prevent evaporation. The cells were w ashed three times w ith either HBSS or PBS and a species-specific biotinylated anti-im m unoglobulin w as then ad d e d (1:50 dilution) for 30 minutes. The cells were w ashed again x3 w ith HBSS/PBS, and a fluorochrom e biotin-streptavidin complex was finally (1:50 dilution) added for 15 m inutes. A fu rther three w ashes followed w ith HBSS or PBS. Fluorescence-labelled cells w ere additionally very briefly im m ersed in propidium iodide dissolved in HBSS (40|Xg/ml) betw een the final w ashes to give the nuclei a red counterstain. The cells w ere m ounted in Citifluor aqueous m o u n tan t an d exam ined using a Zeiss Axiostop routine microscope incorporating reflected light fluorescence w ith appropriate excitation and barrier filters. The cells w ere exam ined at either x40 or xlOO magnification, under oil immersion.