Chapter Two: Materials and Methods
2.4 Other techniques
Phase contrast microscopy
Phase contrast pictures of cell were taken using an inverted microscope (Leica) and the images were processed using Openlab software.
Immunofluorescence
DRG- Schw ann c ell/fib ro b last co-cultures w ere fixed on co v erslip s w ith 4% paraformaldehyde for 30 minutes before washing carefully in PBSA. An acid alcohol (5% acetic acid in m ethanol for 10 m inutes at -20°C) step was included to m aintain neurofilament structure and improve antibody binding. Anti-RT97 antibody (a gift from Dr. John W ood, see Table 2.5) was diluted to 1:1000 in 0.1% BSA, 0.1% azide, PBS. 80pl was added to each coverslip (apart from the secondary controls) and incubated at room temperature in the dark for Ihr. After each treatment the coverslips were washed nine times in PBSA and the relevant secondary was incubated with the coverslips for 45 minutes. The cells were then permeabilised with ice -cold methanol for 10 minutes at -20°C and washed in PBSA before incubating with 1:400 anti -S I00 antibody for Ihr. To amplify the S-100 signal anti- rabbit biotin, (1:100), was added for 30 minutes followed by streptavidin- flouescein, (1:100) for 30 minutes. Hoescht was used at 1:6000 for 20 minutes to detect the cell nuclei. The coverslips were then fixed with ice cold 5% acetic acid in ethanol for 10 minutes at -20°C, mounted using C itifluor (Citifluor) and sealed with nail varnish. Coverslips were visualised using a Zeiss Axioskop microscope with Openlab software.
This protocol was adapted for anti -T hy-1 staining of fibroblasts. Cells were fixed for 10 minutes only in the absence of DRG. See Table 2.5 for the complete list of antibodies used for immunofluorescence and Western blotting.
Staining cells for P-galactosidase activity
Schwann cells were transfected using Fugene 6 reagent as described above and fixed in 4%
paraformaldehyde for 20 minutes at room temperature. The cells were rinsed three times with PBSA and incubated in staining solution (5mM KgFeCCN)^, 5mM K4Fe(CN)6.3H2 0 ,
2mM M gCl2 in PBSA with 1 mg/ml X-gal) overnight at 37°C. The number of blue cells in a
number of fields per dish were counted using an inverted microscope and used to equalise the efficiency of transfection across dishes.
Protein extraction
Cells were treated with Tmx, UV, dibutyryl cAMP or control solvents as described in the results chapters. The cells were washed in IX PBSA and scraped off the dish in 1ml PBSA with a rubber bung. Cells were collected using a chilled microfuge (Heraus) at 13,00rpm and either frozen in liquid nitrogen for later use or lysed in IN or RIPA buffer (Table 2.6), depending on the protein(s) of interest. The cell debris was collected by centrifugation and the protein content of the sample determined using a Bradford assay (BioRad) at OD 595nm. Sample buffer was added to equal amounts of protein and boiled for 5 minutes, prior to separation by SDS-PAGB. 15% gels were used for small proteins (up to about 30kDa), with periaxin requiring a 7.5% gel and phospho -E R K l/2 and a - tubulin being separated on 1 0% gels.
Periaxin is a large protein that is easily degraded. To look at periaxin protein levels in my experiments I harvested the cells in 200|xl of sample buffer directly and boiled the samples at 95°C before storing at -20°C. The samples were disrupted by sonication (Branson sonifier) and the cellular debris removed before use. An equal volume of each sample was separated on a mini protein gel and the levels of a-tubulin determined by W estern blotting
to allow equalisation of protein concentration across samples. The equalised samples were then separated on a maxi protein gel and analysed by Western blotting as described below.
Antibody Origin of antibody Dilution Development of signal
Cyclin D1 Santa Cruz 1/1000 Anti-mouse HRP, PB ST, ECL plus
p l5 Santa Cruz 1/1000 Anti-goat HRP, TBST, ECL plus
pl6lNK4a Santa Cruz 1/200 Anti-rabbit HRP, TBST, ECL plus
p i 8 Santa Cruz 1/200 Anti-rabbit HRP, PB ST, ECL plus
p l9 Santa Cruz 1/200 Anti-mouse HRP, PB ST, ECL plus
p21 Santa Cmz 1/1000 Anti-goat HRP, PB ST, ECL plus
p27 Santa Cruz 1/1000 Anti-mouse HRP, PB ST, ECL plus
piqARP AbCAM for our lab. 1/1000 Anti-rabbit HRP, PB ST, ECL plus
p53 (421) Calbiochem 1/20 Anti-mouse HRP, PBST, ECL plus
PO Gift from J.Archelos 1/1000 Anti-mouse HRP, PBST, ECL normal
Periaxin Gift from P.Brophy,
Edinburgh University
1/30,000 Anti-rabbit HRP, PBST, ECL normal
Oct-6 Gift from D. Meijer,
Erasmus University
1/200 Anti-rabbit HRP, PBST, ECL plus
EGFP AbCAM 1/500 PBST, ECL normal
Raf Santa Cruz 1/400 Anti-rabbit HRP, PBST, ECL normal
ER Santa Cmz 1/500 Anti-rabbit HRP, PBST, ECL normal
P-E R K l/2 Sigma 1/5000 Anti-mouse HRP, TBST, ECL plus
a-tubulin Sigma 1/60,000 Anti-mouse HRP, PBST, ECL normal
SlOO Dako 1/400 Anti-rabbit biotin
Neurofilament (RT97)
G ift from J. W oods, UCL 1/1000 Anti-mouse Cy3 Anti-rabbit HRP Amersham 1/2000 Anti-mouse HRP Amersham 1/2000 Anti-goat HRP Dako 1/2000 Streptavidin- fluorescein Amersham 1/100 Anti-rabbit Biotin Amersham 1/100 Anti-mouse Cy3 Jackson Immunological research labs 1/1000
Table 2.5. Antibodies used for Western blotting and immunofluorescence.
Solutions Components and additional information
IN lysis buffer 1% N P-40, 150mM NaCl, 50mM Tris pH 8 , 20mM NaF,
lOOpg/ml PMSF, 15|ig/ml aprotinin and ImM Na^VO^. RIPA lysis buffer
(Use BioRad protein assay)
1% Triton X-100, 0.5% Sodium deoxycholate, 5GmM Tris pH7.5, lOOmM NaCl, ImM EGTA pHS, 20mM NaF, lOOpg/ml PMSF, 15|ig/ml aprotinin, ImM Na^VO^.
4X Sample buffer 200mM Tris pH 6 .8, 8% SDS (BioRad), 40% glycerol, 400mM
DTT, 0.25% bromophenol blue.
lOX Running buffer 2.5M glycine (BDH), 250mM Tris, 1% SDS. lOX Transfer buffer 200mM Tris, 1.5M glycine, 20% methanol (BDH) Milk block 5% milk, 0.05% Tween -20(BioRad) in PBS Stripping Buffer 200mM glycine , pH2.5, 0.4% SDS
IX PBSA Tween wash 0.05% Tween-20 in PBSA
20x TBS Tween 200mM Tris, pH8, 3M NaCl 1% Tween -20
PBSA 137mM NaCl, 2.7mM KCl, 1.47mM KH^PO^, 8 .ImM Na^HPO^
20X TBS 200mM Tris pH 8, 3M NaCl.
Table 2.6 Solutions for protein extraction and Western blotting
Western Blotting
Proteins were transferred to PVDF membrane (Immobilon P, M illipore) using transfer apparatus (see Table 2.6). Membranes were blocked overnight at 4°C or for 45 minutes at room temperature. Blots that were being probed for periaxin protein levels were blocked overnight with a modified block solution consisting of 1% milk (Merck), 0.05% Tween -2 0 in PBSA. (Blots for re-probing were washed in stripping buffer for 30 minutes and then blocked in milk.) Membranes were incubated in primary antibody in plastic bags at room tem perature on a rocking platform (Stuart Scientific). The antibody was diluted in the remaining block to the required concentration (see Table 2.5). A fter Ihr the blot was washed in either PBST or TBST (higher stringency) solutions for three sets of 5 minutes. The relevant secondary antibody was incubated with the blots for Ihr and the washing stage
repeated. A final wash was made in PBS or TBS alone and the blot half-dried using W hatman 3MM paper. ECL solution (ECL normal or plus depending on the antibody, Amersham) was carefully pipetted onto the protein side of the blot and incubated at room temperature for 5 minutes. After developing the blot was dried as before and wrapped in cling film. The blot was exposed to film (Biomax ML, Kodak) for the required time in a dark room and the films developed using an Agfa automatic film processor.
Statistical analysis
Results were analysed using the student’s two-sample T-test to test whether measurements made on two populations were different from each other (Ennos, 2000). The null hypothesis proposed that the two populations were the same in every case.