2.4.1 Fixation and labelling of lysosomal membrane protein lgp and ryanodine receptor subtypes 1, 2 and 3 in isolated pulmonary artery smooth muscle cells
Sequence specific antibodies to the RyR subtypes 1, 2, and 3 were a gift from Professor Sidney Fleischer and have previously been shown to be highly sequence specific for their individual targets on the different RyR subtypes (Lesh, et al., 1992; Jeyakumar, et al., 1998; Jeyakumar, et al., 2001); Table 2.1). GM10 antibodies (Grimaldi, et al., 1987), raised against the lysosomal glycoprotein antigen lgp120 (Lewis, et al., 1985) were a gift from Professor
preferentially associated with the membrane of lysosomes (Lewis, et al., 1985; Howe, et al., 1988). Antibody Raised in Dilution Immunocyt. Dilution
Westerns Target sequence References
RyR1 Rabbit 1:500 1:500 Residues 4476-4486 Leshet al, 1993 RyR2 Rabbit 1:500 1:500 Residues 1344-1365 Jeyakumaret al, 2001 RyR3 Rabbit 1:500 1:500 Residues 4236-4336 Jeyakumaret al, 1998
Table 2.1: Dilution factors, sequence specifics and initial references relating to anti- ryanodine receptor (RyR) antibodies for immunocytochemistry (Immunocyt.) and Western blotting (Westerns) investigations.
200l of freshly dissociated cell suspension was put onto poly-d-lysine coated glass coverslips and cells were allowed to settle for 1 hour at room temperature (~22 oC). Coverslips were covered in Poly-D-lysine to coat the surface with a net negative charge in order to enhance cell adhesion. After 1 hour, excess solution was removed and the coverslips placed in freezing cold methanol for 15 minutes to fix the cells. Methanol fixation works by removing lipids and dehydrating the cell while denaturing and precipitating proteins on the cellular architecture. This method allows for quick fixation of the cells; however it can result in the loss of diffuse proteins, i.e. those proteins which are not anchored to membranes and are free to move within the cytoplasm of the cell. Once fixation was complete cells were removed from the methanol and were permeabilised by three, five minute washes in phosphate buffered saline (PBS; pH 7.4) containing the non-ionic detergent Triton X-100 (polyethyrene glycol mono-p-iso-octylphenyl ether; 0.3%). Triton X-100 causes the formation of pores in the membranes of cells, thus allowing access of specific antibodies to the inside of the cell. After this cells were washed 3 x 5 minutes with blocking solution (1% bovine serum albumin (BSA), 4% goat serum and 0.1% Triton X-100). Blocking solution contains goat serum and BSA to prevent any non- specific binding of the primary antibodies within the cell. This significantly improves signal to noise ratio within the sample. Triton X-100 is included in the solution to aid the penetration of goat serum, BSA and primary antibodies. Cells were then incubated overnight at 4oC with antibodies raised in rabbit to sequences of one of the RyR subtypes and with GM10
antibodies. These antibodies were diluted in blocking solution to help minimize non specific binding (Table 2.1).
The following day cells were washed three times for five minutes in fresh blocking solution of the same composition of that used previously. In order to determine where the primary antibody has bound within the cell it is necessary to employ some means of being able to visualize it. This was achieved by employing a secondary antibody raised against immunoglobulins of the species that the primary antibody is raised in. Secondary antibodies are conjugated to a fluorescent molecule, allowing for visualisation. Following the washing step, cells were incubated for two hours in the dark at room temperature (~20 oC) with fluorescently labelled secondary antibodies. In order to visualize the primary antibodies raised in rabbit against RyRs an anti-rabbit antibody raised in goat was used. This antibody was conjugated with the red fluorescent dye Texas Red (excitation 555 nm, emission 617 nm) and diluted 1:200 with blocking solution. The primary antibody, GM10, raised against the lysosomal membrane glycoprotein lgp120, was visualised using an anti- mouse secondary antibody raised in goat and conjugated to the green fluorescent dye FITC (excitation 490 nm, emission 528nm; diluted 1:200 with blocking solution). These two dyes are suitable to use for co-labelling studies as their excitation and emission spectra show very little overlap (Fig. 2.4).
Fig. 2.4Diagram of excitation and emission spectra for the fluorescent probes FITC and Texas Red showing the lack of overlap between the two
Cells were washed five times for five minutes each in the dark with fresh PBS (pH 7.4) following the two hour incubation. Cells were then attached
300 400 500 600 700 Wavelength (nm) F lu o re s c e n c e in te n s it y
excitation emission excitation emission
to microscope slides using hard setting, anti-fade mountant containing the fluorescent nuclear dye 4’6-diamidino-2-phenylindole (DAPI; excitation 360 nm, emission 457 nm). DAPI has been shown to associate with the minor groove of double stranded DNA, preferentially binding to AT clusters (Kubista, et al., 1987). The binding of DAPI to the minor groove produces around a 20 fold increase in fluorescence under excitation allowing for accurate visualization of the nuclear region of the cell (Barcellona, et al., 1990).
The slides were then left for 2 hours in the dark at room temperature (20oC) to allow the mountant to set. Slides were then stored in the dark at 4oC until examined.
2.4.2 Preparation of control slides for use in immunocytochemical investigations
In order to remove background fluorescence and allow for examination of the binding of secondary antibodies within cells, two sets of control slides were prepared in parallel to test slides.
The first control slides differed from test slides in that these control slides were not incubated with primary antibodies overnight. This allowed me to examine the fluorescence intensity of background fluorescence associated with the presence of secondary antibodies within the fixed cells (Fig. 2.5A). By determining the level of intensity of secondary antibody fluorescence in control slides, following deconvolution using Softworx software (Chapter 2, Section 2.3.4), it was possible to determine a ‘threshold’ fluorescence intensity level in order to remove background fluorescence from test slides, enabling visualization of specific labelling within the test cells (Fig. 2.5B).
The second set of control slides were prepared to confirm the specificity of the primary antibodies by incubating a given concentration of primary antibodies with a 10X concentration of the specific sequence the primary antibodies are raised against. Following three hours of incubation of the primary antibodies with their ‘blocking peptide’, slides were prepared as described above. The fluorescence intensity of these control slides were then examined for comparison against test slides. The absence of fluorescent
labelling above the ‘threshold’ fluorescence intensities determined from control slides prepared in the absence of primary antibody confirmed the specificity of primary antibodies.
Fig. 2.5:Elimination of background fluorescence from images obtained on the Deltavision system:Panel A(i)shows a deconvolved Z-section (depth 0.28m) taken through an isolated pulmonary artery smooth muscle cell from a control slide not incubated with primary antibody against RyRs or lysosomes. The cell has been incubated with fluorescent-conjugated secondary antibody (red/green) and is labelled to show the position of the nucleus (blue). A(ii)shows a deconvolved Z-section (depth 0.28 m) through a different isolated pulmonary artery smooth muscle cell that was incubated with primary antibody against RyRs or lysosomes. Binding of fluorescent-conjugated secondary antibodies shows the distribution of RyR3 (Red) and the lysosomes (green), although considerable background fluorescence is still seen. The position of the nucleus is indicated in blue.Panel B(i)shows the same cell as inA(i)following adjustment to remove background fluorescence, the position of the nucleus is still apparent (blue). B(ii) shows the same cell as in A(i) following adjustment to remove background fluorescence, the distribution of RyRs (red) and lysosomes (green) can be easily determined in relation to the position of the nucleus (blue).
2.4.3 Visualisation of labelling in methanol-fixed, isolated pulmonary artery smooth muscle cells
Fluorescent labelling in methanol-fixed, isolated pulmonary artery smooth muscle cells were visualised using the Applied Precision Deltavision
were captured at 0.2 m steps in the Z-direction. Captured images were deconvolved using the method of deconvolution contained within the Deltavision software, described in Chapter 2, Section 2.3.4. Volumetric analysis of fluorescent labelling was carried out using Volocity Software (Improvision, UK).
2.5 Detection of proteins in pulmonary artery smooth muscle by use of