2.7 NEXEDGE
2.7.1.9 Software
DRGs were fixed, detergent-permeabilised, incubated with the affinity purified antipeptide anti- G aj antibody emd prepared for immunocytochemistry. G aj immunostaining was associated with the inner plasma membrane (Figure 3.27 C-J), weak immunoreactivity was also observed in the neurites (Figure 3.27 A-G) and cytoplasm (Figure 3.28 A).
Preincubation o f the anti-Gaj antibody with its immunising peptide (50 pg/m l Ih at 37^C] abolished specific G aj immunoreactivity (Figure 3.28 B). The control rabbit IgG (50 p g /m f produced no discernible immunostaining (Figure 3.28 C).
3.4.2.3 A n alysis o f the loss o f Gclq a n d (7a/ im m u n oreactivity follow in g prea d so rp tio n o f th e anti-GcL^ an d anti-GoLQ antibodies with th eir respective im m unising peptides.
The degree o f G ao G aj immunoreactivity associated with each DRG neurone wa; measured using the image analysis facility supplied with the confocal microscope. A cursor wai used to outline the fluorescent region associated with each cell, and using the corresponding phase contrast image to differentiate the boundary o f the cell, an average grey value intensity (GVI) value was ascribed to each DRG. GVI values were obtained for cells with had beei incubated with the affinity purified anti-Gao (Figure 3.29 A ) and anti-Gaj (Figure 3.29 B antibodies, either in the presence or absence o f their respective immunising peptides (50 pg/ml
Ih at 37°C ). Preadsorption o f anti-Gao antibody with its immunising peptide reduced Gœq
immunoreactivity to the level o f immunofluorescence observed follow ing treatment o f the cells with control rabbit IgG (50 pg/ml) (Figure 3.29 A). Similarly, preadsorption o f anti-Gaj antibody with its immunising peptide, reduced G aj immunoreactivity to the level o f immunofluorescence observed following treatment o f the cells with control rabbit IgG (50 p g/m l) (Figure 3.29 B)
U) UJ
Figure 3.24 Immunolocalisation of Gttg throughout the z-plane o f DRG neurones
DRG neurones were fixed with 4% paraformaldehyde, detergent-permeabilised with 0.2% Triton-XlOO, incubated with anti-G «0 antiserum (OC2-Provided by Professor Milligan, Glasgow University) and prepared for immunocytochemistry.
A series of 2 pm confocal sections (A-L) taken through the z-plane of the cell from the uppermost surface of the cell (A) to the region of cell-attachment (L). G ag immunoreactivity was prevalent at the inner surface of the plasma membrane (pm), in the neurites (n) and within cytoplasmic regions (c).
A 2 pm section taken 10 pm up from the attachment plaque in a cell incubated with the corresponding preimmune serum (M) failed to produce any immunoreactivity. Scale bar = 25 pm.
U)
Figure 3.25 Immunolocalisation of G ag throughout the z-plane of DRG neurones
DRG neurones were fixed with 4% paraformaldehyde, detergent-permeabilised with 0.02% Triton-XlOO, incubated with the affinity purified anti-Gag antibody and prepared for immunocytochemistry.
A series of 2 pm confocal sections (A-J) taken through the z-plane of the cell from the uppermost surface o f the cell (A) to the region o f cell-attachment (J).
G ag immunoreactivity was prevalent at the inner surface of the plasma membrane (pm), in the neurites (n) and at the attachment plaque (ap).
psW
F igu re 3.26 Im m u n olocalisation o f G a o
D RG neurones w ere fixed w ith 4% paraform aldehyde, detergent-perm eabilised w ith 0.02% Triton-XlOO and incubated w ith the affinity-purified an ti-G ao antibody (50 pg/m l).
A, im m unostaining associated w ith Guq w as observed around the inner plasm a m em brane and at regions o f cell-to-cell contact.
B, preadsorption o f the a n ti-G a o antibody w ith its im m unising peptide (50 pg /m l, Ih at 30 °C ) ab olished G a o im m unoreactivity.
C, control rabbit IgG (50 pg /m l) failed to produce any im m unofluorescent signal.
F luorescent im ages are 2 p m sections taken m idw ay through the cell. The corresponding phase contrast im ages are on the right. Scale bar = 20 pm .
U)
Os
Figure 3.27 Immunolocalisation of G ttj throughout the z-plane of DRG neurones
DRG neurones were fixed with 4% paraformaldehyde, detergent-permeabilised with 0.02% Triton-XlOO and incubated with the affinity-purified anti-G aj antibody (50 pg/ml).
A series of 2 \im confocal sections (A-K) taken through the z-plane of the cells from the top surface of the cell (A) to the region o f cell-attachment (K). The corresponding phase contrast image (L) is also shown.
G aj immunoreactivity was revealed primarily around the inner plasma membrane (ipm), although some G a; immunoreactivity was also detected in neurites (n). Weak G aj immunoreactivity was also observed in the cytoplasm (c). Scale bar = 25 pm.
F igure 3.28 G a j im m unoreactivity w as abolished by preadsorption o f the affinity-purified a n ti-G a j antibody w ith its im m unising peptide
D R G neurones w ere fixed w ith 4% paraform aldehyde, detergent-perm eabilised and incubated w ith the affinity-purified a n ti-G a j antibody (50 pg/m l).
A, im m unostaining asssssociated w ith G a j w as observed around the inner plasm a m em brane. B, preadso rp tion o f the a n ti-G a j antibody w ith its im m unising peptide (50 pg/m l Ih at 37®C) ab olished G a j im m unoreactivity.
C, in cu bation o f the cells w ith control rabbit îgG (50 pg/m l) produced no im m unofluorescent signal.
F luo rescen t im ages are 2 p m confocal sections taken m idw ay through the cell. The correspo nd ing phase contrast im ages are on the right. Scale bar = 20 pm .
A
G a,B
Ga.g
I
Ü 140 -n 120 - 100 - 80 - 60 — 40 - fe 2 0 - 01
TI
I
160 -1 140 120 100 80 60 40 20 0 * JL Anti-GttQ antibody (50 p.g/ml)Anti-Gao antibody + immunising peptide (50 pg/ml)
ESS Control rabbit IgG (50 pg/ml)
■■ Anti-Gaj antibody (50 pg/ml)
I— ' Anti-Gaj antibody + immunising peptide (50 p.g/ml)
tSS3 Control rabbit IgG (50 pg/ml)
F igu re
3.29
A nalysis o f thereduction
inGUq
andGai
im m unoreactivity follow in g preadsorption o f the antibodies with their respective im m unisin g peptidesThe level o f G a o and G aj immunoreactivity associated with each DRG neurone w as measured using the image analysis facility supplied with the confocal microscope. A cursor was used to outline the region o f immunofluorescence associated with each cell, using the corresponding phase contrast image to mark the boundary o f the cell. An average grey value intensity (GVI- arbitary units) was then ascribed to each cell.
A, cells were incubated in either the anti-Gao anti serum (■■■) or anti-Gao antiserum which had been preincubated with the corresponding immunising peptide (50 pg/m l, Ih at 37®C)
(C U D ). The average GVI value for cells incubated with control rabbit IgG (50 |ig/m l) is also shown ( R ^ ) . Results are expressed as mean ± sem. *p<0.05 compared to the GVI o f cells incubated with anti-G ao antiserum in the absence o f immunising peptide, n=8.
B, cells were incubated in either the anti-Gaj antiserum (■■■) or anti-Gaj antiserum which had been preincubated with the corresponding immunising peptide (50 |ig/m l, Ih at 37®C) (I 1).
The average GVI value for cells incubated with control rabbit IgG (50 pg/m l) is also shown ( R ^ ) . Results are expressed as mean ± sem. *p<0.05 compared to the GVI o f cells incubated with anti-Gaj zmtiserum in the absence o f immunising peptide, n=5.