4.4 PRUEBAS PARA DETERMINAR EL EFECTO DE INHIBICIÓN SOBRE
4.4.2 PRUEBA DE AZUL DE METILENO (MBT)
4.4.2.1 Fluido Antes de Rolar
Preparation of DALd plasmid template
F G F R l exon Ilia, FGFR2exon Illy (KGFR) and FGFR2 exon IIIc (BEK),w ere am plified from norm al hum an genom ic D N A and cloned in to Bluescript II SK"*" (Chan and T horogood, 1999). T h e identities o f the FG FRexons were verified by sequencing. IM A G E C onsortium cD N A clone (G enB ank N o T54933), corresponding to hum an osteonectin was obtained from the M edical Research Council H um an G enom e M apping Project R esource C entre (H inxton, Cam bs, U IQ and subcloned into B luescript II SK+.
D N A plasm ids were then prepared from colonies o f stock attenuated E Coli, containing the specific cloned Bluescript II 8 1 0 vectors. T hese w ere stored at -70 °C, and aliquots as required w ere grow n overnight in LB b ro th on an aerating h o tplate at 37 °C.
T he overnight culture was centrifuged at ISOOrev for 10 m inutes in 50 m l F alcon tubes, and pellets w ere resuspended in 5 mis Solution 1 (A ppendix C) by vortex.
Solution 2 (5 mis. A ppendix C) was added, and m ixed by inverting to clarity o f the resulting hom ogeneous solution, w hereupon Solution 3 (5 mis. A ppendix C) was added, and m ixed by inverting until a fine w hite protein precipitate appeared. This precipitate was centrifuged, and the su p ernatant rem oved.
A n equal volum e o f isopropanol was added to the supernatant, m ixed thoroughly and centrifuged. T h e resulting pellet (protein and nucleic acid) was resuspended in distilled w ater and aHquoted into ep p e n d o rf tubes.
Equal volum es o f phenol: chloroform : resuspension w ere m ixed in solution in the e p p e n d o rf tube by vortex, and centrifuged. T h e u p p er phase was rem oved to fresh e p p e n d o rf tubes, 1 m l o f ice cold 100% ethanol added and the m ixture centrifuged.
T h e pellet (plasmid D N A , bacterial nucleic acid) was w ashed in 70% ethanol, and resupended in 50pl T E w ith 5pi R N ase at lO m g/m l.
L ithium chloride 50pl was added, the solution m ixed thoroughly, and kept on ice for 10 m inutes.
T h e solution was centrifuged, and the supernatant containing plasm id D N A retained. T w o parts 100% ethanol w ere added, and the solution centrifuged aggressively.
T h e pellet, containing plasm id D N A , was w ashed in 70% ethanol, resuspended in 50pl T E and stored at -20C.
Estimation of plasmid concentation
A 1 pi aliquot was serially diluted xlO, and concentration was estim ated by electrophoresis on a 0.8% agarose gel (Fi^ 2.2.2-1).
Unearising Plasmids to form a D N A . template
Plasm id D N A containing the tem plate sequence for rib o p ro b e synthesis was linearised with the appropriate D N A ligating enzyme in a total reaction volum e o f SOOpl as follows:
L in e a r is a tio n m i x ; T o ta l v o lu m e SOOjul
D E P C H 2 0 410 111
lOx E nzym e B uffer 50pl
Plasm id D N A 30pl ( ip l/p g )
R N A ’se (lO pg/m l) 5pl
Enzym e:
F G F R l plasmid: H indi III
Osteonectin plasmid: H indi III
F G F R 2 — bek plasmid: X b a l
K G F R plasmid: X b a l
T h e reaction volum e was m aintained at 37 °C for one hour, after w hich 10|Xl o f the linearised D N A was quality assessed by gel electrophoresis (Fig 2.2.2-2).
T h e linearised D N A tem plate was then further subjected to phenol: ch loroform extraction, in an equi-volum e solution w ith the linearisation mix.
T h e D N A : phenol: chloroform solution was vortex m ixed and centrifuged. T h e u p p e r phase, containing the linearised D N A tem plate, was m ixed by careful pipette w ith 2 volum es o f 100% ethanol, and 1 /1 0 * volum e 3 m olar sodium acetate.
T h e resulting solution was m ixed thoroughly, and centrifuged, to bring dow n a D N A pellet, w hich was resuspended in 20)Lll D E P C H 20 and stored at —20 °C.
Fiboprobe synthesis and purification
T h e follow ing procedures were undertaken in a designated laboratory for radioactive
substances. All laboratory hardw are used for these procedures was rendered TIN A grade’ by autoclave at 180 °C to destroy am bient R N A ’se, and aU solutions, w here possible, were autoclaved w ith D E P C to achieve the same effect.
Linearised D N A tem plate and R N A nucleotides (35S-UTP) w ere in tro d u ced w ith the
appropriate R N A polym erase in a total reaction volum e o f 25pl, and incubated at 37 °C under a m ineral oil meniscus:
T ra n scrip tio n R ea ctio n v o lu m e 25pl
D E P C H 2 0 7.5pl
5x T ran scription buffer 5pl
D T P O.IM 2.5 pi
N ucleotide G A C mix: (equivolume am ounts o f lO m M A T P , lOm M O T P , lOm M CTP)
3pl
35S - U T P (lOOpCi) 5pl
D N A Linearised plasm id V I (V g )
R N A guard O.Spl
T h e rib o p ro b e was eluted through a swollen sephadex G 50 bead colum n to bring dow n the transcribed riboprobe. A 1ml drip colum n was designed in a suitable P asteur pipette, and rinsed three times w ith 300pi elution buffer.
T h e transcription reaction volum e was aspirated from u n der the m ineral oil and loaded into the colum n; then eluted w ith ISOpl elution buffer. T h e colum n was further eluted three times w ith 150 p i elution buffer, allowing each eluent to drain through p rio r to adding the next, and this first com bined eluent was discarded.
A series o f ep p e n d o rf m bes was set up in a rack, and the elution colum n - Pasteur pipette m oved to the first o f these. ISOpl elution buffer was added to the top o f the colum n and allowed to drain through it completely, to be collected in the first e p p e n d o rf tube. A fter it had com pletely drained, the colum n was m oved to the second eppendorf, and a second elution repeated.
T h e colum n was eluted a total o f six times, such that the eluted p ro b e — transcription reaction was collected in a series o f six ep p e n d o rf tubes in series.
I p l o f each eluted fraction was placed in a series o f wells to w hich w ere added 40 pi scintillation fluid. T he beta - radioactive em ission was counted on a calibrated WaUac
scinticounter and displayed graphically and numerically using M icrobeta softw are 2.2.2-J).
T h e optim um spectrum is a peak em ission o f the two ‘b est’ fractions at the apex o f a bell — shape distribution. T he two ‘b est’ fractions represent the fractions containing the synthesised riboprobe. T h e rem aining samples represent beta em issions from p ro b e fragm ents and scattered nucleotides and w ere discarded.
T h e ‘b est’ fractions o f ISOpl each w ere com bined, and 2 p i (20pg) o f yeast R N A (lO m g/m l) was added. T o this solution, 30pl 3M lithium chloride was added and the solution
precipitated w ith 900 p i ethanol, after m ixing thoroughly and incubation over dry ice for 20 m inutes.
T h e pellet (riboprobe) was w ashed in 70% ethanol, and dissolved in 50 m l hybridisation mix, w ith the solution then m ade up to 10’5 c p m /p i and used im m ediately o r stored at —20 °C.
IPreparation o f slides and in - situ hybridisation
Sections, m o u n ted on T E S P A subbed slides (A ppendix B), w ere dew axed by im m ersion in two fresh sequential H istoclear (Fissons) solutions for 10 m inutes each, and th en dried in 100% ethanol by sequential im m ersion in two fresh solutions for 10 m inutes each.
Slides w ere then rehydrated by transfer through a sequentially less concentrated ethanol series for 2-3 m inutes each (E th a n o l/ D E P C w ater 95% - 85% - 70% - 50% - 30%)
Slides w ere sequentially w ashed in saline, then PBS, for 5 m inutes each, and prefixed in 4% PFA at ro o m tem perature for 20 m inutes. Tw o further PBS washes w ere perfo rm ed for 2 m inutes each.
Slides w ere treated w ith lO pg/m l Proteinase K in PBS for 5-10 m ins only, and w ashed in in PBS for 5 m inutes. Slides were then p o st fixed in 4% P F A for 5 m inutes, and w ashed in D E P C w ater for a further 5 m inutes.
Slides w ere treated w ith acetic anhydride in a fum e cupboard. 400 mis o f O.IM
triethanolam ine w ere b ro u g h t to p H 8 w ith sodium hydroxide, into w hich the slides w ere im m ersed, and acetic acid added dropw ise to a constantly stirred solution.
Slides w ere again w ashed in PBS and saline for 5 m inutes each, th en dehydrated by transferring through an alchohol series (E thanol 30% - 50% - 70% - 85% - 95% - 100%- 100%; 2-3 m inutes each).
Slides w ere then air dried in a fum e cupboard and the p ro b e in tro d u ced for hybridisation on the same day.
Hybridisation
T he hybridisation steps w ere conducted in a specially designated and separated laboratory for radioactive chemicals.
T he p ro b e in hybridisation m ix was b ro ught to 80 °C for 2 m inutes on a preheated h o t plate, then cooled to am bient tem perature.
H ybridisation m ix was applied to each se ctio n /se t o f sections on the prep ared slides
according to the volum e required for adequate cover (15 pi o f hybridisation m ix p er 2 2 / mm^ ), and so as to avoid bubbles under the silicone cover sUp.
T h e hybridising sections o n each slide were placed horizontally in a black hybridisation box together w ith a piece o f tissue soaked in 10 mis 50% form am ide and 5x SSC in the b a c k /b a se to prev en t slides drying out.
T h e hybridisation box was sealed w ith tape, placed in plastic bag, and sections left to hybridise overnight, weighed dow n in a 55-60 °C w ater bath.
Posi hybridisation treatment (ùr slide washing
Racked slides were soaked in 5x SSC and 10 m M D T T at 55C for 30 m inutes, and the cover slips encouraged to separate by gentle agitation. Studies involving suture m aterial w ere co nducted w ith particular care. This w ash was repeated.
Slides w ere then washed in form am ide w ash solution at 65 °C for 30 m ins.
Slides w ere w ashed x3 in T E N buffer at 37 °C for 10 m inutes, to w ash away form am ide. T he slides w ere then treated w ith lO p g /m l R N A ’se A in T E N b u ffer at 37 °C for 30 m inutes to digest rem aining unhybridised probe.
Slides w ere then w ashed w ith T E N buffer at 37 °C for 15 m inutes.
Slides w ere w ashed again in form am ide w ash at 65 for 30 m inutes.
Slides w ere th en sequentially w ashed w ith 2x SSC, and O.lx SSC fo r 15 m inutes.
Slides w ere then dehydrated by sequential w ashing through an a lch o h o l/0 .3 M am m onium acetate series (30% - 60% - 80% - 95%) and finally in absolute alchohol, p rio r to air drying in a fum e cupboard.
A.utoradiography
Blue silica gel was w rapped in tissue paper and taped to the inside o f a dark box, to act as a dessicant environm ent.
7.5 mis em ulsion (K4, Ilford Com pany) was m elted at 43 °C in a slide mailer, and an equal volum e o f prew arm ed 2% glycerol was added.
Shdes were dipped into the em ulsion mix, laid o u t to dry, and placed in the b o x containing dessicant. T h e box was closed tightly, and w rapped in tin foil. Shdes w ere left to expose for 3- 7 days, depending u p o n the p ro b e in use and the tissue type, incubated at 4 °C. T e st shdes w ere developed p rior to developing the w hole series to assess optim um tim e fo r incubation.
T h e incubation period o f the hybridised sections pro v ed to be a function o f the age and type o f the probe. TSfewer’ probes were those m ade closer to the activity date o f the ^^S isotope and required shorter incubation periods. T he osteonectin p ro b e had the sh o rtest exposure tim es, and the F G F K p ro b e exposure times w ere variable. In practice, test shdes were developed for each series o f studies at time periods from 3 — 7 days. AU p robes were optimaUy developed on the tissue sections by this time.
Developing and staining of slides
T h e shde box was rem oved from 4 and b ro ught to ro o m tem perature.
Shdes w ere rem oved from the box in dark ro o m environm ent, and passed th ro u g h developing solution (2 m ins), 1% acetic a c id /1% glycerol (1 m in), and fixative solution (2 mins).
Shdes w ere then w ashed in w ater for 10 m inutes, subsequendy stained w ith toluidine blue for 10 m inutes, and rinsed twice in w ater p rio r to destaining.
Shdes w ere destained in an alch o h o l/0 .3 M am m onium acetate series o f increasing concentration (30% - 50% - 70% - 85%) then in alchohol (95% - 100%).
C over shps (washed in 100% alchohol) w ere m o u n te d on the sections using D P X m o u n tan t, taking care to exclude bubbles. T he shdes w ere left to dry p rio r to dark field m icroscopy.
2.2.3 Immunohistochemistry fo r protein detection in
-situ
Introduction
In parallel to the analysis o f F G F R transcript expression by the i/z - situ hybridisation technique, the detection o f the F G F R p rotein p ro d u ct was undertaken by im m uno - histochem istry. C om parison o f the data achieved by each technique substantiates the o th er and may give an insight into m olecular events betw een transcript expression and the detection o f the protein p ro d u ct in - situ.
Im m unohistochem istry is a routine histochem ical technique. F o r conventional
im m unohistochem istry using tissue sections, tissues are first fixed to preserve m orphology, then dehydrated, em bedded in a wax block and cut into thin (3-6 pm ) slices. T hese are placed on a glass shde, w hich has been pre-coated to facihtate adhesion. Sections are then re hydrated, m ay be pre-treated to increase penetration o f the antibodies as necessary, and then ‘blocked’ w ith serum a n d /o r album in. T his prevents non-specific antibody binding. O ptim al blocking may be obtained w ith serum o f the same species as the secondary antibody, b u t go o d results are obtained in m o st reactions w ith foetal calf serum .
Prim ary antibodies are then apphed. In direct im m unohistochem istry, prim ary antibodies are directly conjugated to a detection system. A lthough this eliminates the non-specific
background signal generated by secondary antibodies, it is less sensitive than indirect
techniques. Indirect techniques have been used th ro u g h o u t this thesis. In these studies, the prim ary antibody is unconjugated, and is detected by a conjugated antibody raised against the prim ary h o st species.
T h e detection system used in this thesis is the avidin - bio tin reaction to a biotinylated secondary antibody. T his m eth o d is based on the ability o f the egg-white glycoprotein avidin to n o n - im m unologically bind four m olecules o f the vitam in biotin, conjugated to the secondary antibody.
M ultiple copies o f the biotinylated secondary antibody bin d w ith the unlabeUed prim ary antibody (bound to the tissue antigen o f choice). T he section is then treated w ith the avidin- biotin peroxidase com plex, w hich again binds m ultiple sites, such th at the signal is greatly amplified. Prim ary antibodies can therefore be used at a high dilution, w hich reduces the false - positive background staining.
A vidin - bio tin peroxidase activity is then detected by a brow n colour change on exposure w ith D A B. A brow n precipitate is produced by the peroxidase in the strep ta vidin-bio tin m ixture, and this can be m o n ito red by direct vision or m icroscopically. A ppropriate counterstaining allows accurate assessm ent o f the p rotein expression dom ains w ithin the histological preparation.
T he prim ary antibodies used in this thesis w ere anti — hum an, raised in a variety o f h o st