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(6 6mM Tris-HCl, 0.66mM MgClz, ImM 2-mercaptoethanol in distilled

water adjusted to pH 7.6) and 100 units of Exo III for 30 minutes at 37°C.

8. 3ml of PBS diluted ten times with distilled water were added, then

centrifuged. The pellet was then ready for suspension in the first antibody.

DNA profiles prepared by this method also contained a large proportion of debris and showed very little positive staining. The lack of positive staining with different dénaturation procedures suggested that there may be deficiencies in the antibody procedures. Thus the protocol producing the best defined DNA profile was selected for further optimisation.

This protocol utilised fixed enzyme digested single cell suspensions and the cells were denatured with pepsin and HCl to expose the antigenic sites (Figure 7.6).

7.2.4 Antibody Incubation

Primary antibodies available for attachment to the proliferative and hypoxic probes consisted of rat or mouse anti-BrdUrd and rabbit anti­ theophylline. Secondary antibodies consisted of FITC conjugates of rat, mouse and rabbit immunoglobulins or biotin conjugates of rat, mouse and rabbit immunoglobulins which could be biochemically linked to streptavidin-R-phycoerythrin or directly to R-phycoerythrin as the second antibody fluorochrome.

The first combination tried was simultaneous incubation with the standard primary antibodies 25pl rat anti-BrdUrd (1 in 10 dilution) and 250pl rabbit anti-theophylline (neat) for 1 hour as they were derived from different species. The weakest primary antibody (rabbit anti-theophylline) was combined with the strongest fluorochrome (goat anti-rabbit FITC) and biotin conjugated anti-rat immunoglobulin at 1 in 1 0 dilutions for 1 hour

(A) 2M HCl for 25 minutes

s e e 4ee

(B) 0.2mg/ ml pepsin in 2M HCl for 10 minutes

le e e s e e

(C) 0.2mg/ ml pepsin in 2M HCl for 20 minutes

l e e e e e e

Figure 7.6 Comparison of DNA profiles from different DNA

dénaturation procedures using SaF tumours. 194

in PNT. Then 1:10 diluted R-phycoerythrin-conjugated streptavidin was incubated with the nuclei for 30 minutes and 7-AAD used as the DNA marker. There was no positive staining for either of the markers with pepsin digested nuclei direct from tumour pieces but positive NITP adduct staining when denatured single cell suspensions were used with the same antibody combination. In all subsequent staining attempts the antibodies were kept at the same dilutions as above, unless otherwise stated, and the

primary and secondary antibodies incubated for 1 hour at room

temperature.

Further manipulations were carried out on the BrdUrd portion of the staining procedure. Double staining of BrdUrd with single cell preparations using the standard rat anti-BrdUrd and FITC conjugated anti-rat antibodies showed evidence of positive staining but the same samples utilising anti-rat biotin with R-phycoerythrin conjugated streptavidin did not produce positive results when either diluted in PNT or theophylline antiserum. The secondary antibodies were then switched so that anti-rat FITC conjugate and anti-rabbit biotin conjugate were used in conjunction with R- phycoerythrin conjugated streptavidin. This produced little positive staining for either investigative probe. The biotin-streptavidin complex may not have produced good results because the large R-phycoerythrin molecule could not penetrate the DNA double helix to attach to the biotin. The binding of R-phycoerythrin to BrdUrd appeared to be the main problem. Therefore in an attempt to simplify this procedure, a direct conjugate of R-phycoerythrin to a mouse IgG was tried. It should be noted that this was not used originally because the signal intensity can be improved using the biotin/streptavidin system, and that the standard BrdUrd antibody was developed in rat and no anti-rat IgG R-phycoerythrin conjugates were available. Therefore the monoclonal was changed to a mouse derived antibody from Dako. Initially this protocol demonstrated no positive staining for BrdUrd so a Caltag mouse monoclonal (BR3), which recognised bromodeoxyuridine specifically, was used in combination with the anti-mouse R-phycoerythrin.

All the procedures tried above failed to provide positive triple staining so it was thought that the two primary antibodies could be blocking each other and thus the two staining processes were tried in succession.

2M HCl dénaturation of fixed single cell suspensions for 25 minutes was followed by different combinations of primary and secondary antibodies. NITP/anti-rabbit biotin conjugate and BrdUrd/anti-rat FITC conjugate failed to provide good NITP staining. NITP/anti-rabbit FITC conjugate and BrdUrd/anti-rat biotin conjugate produced good hypoxic and BrdUrd staining profiles, as did the combination NITP / anti-rabbit FITC conjugate and the direct anti-mouse R-phycoerythrin conjugate for BrdUrd. However, the second positive combination with the direct anti­ mouse R-phycoerythrin conjugate had greater green fluorescence and the calculated values for NITP and BrdUrd staining were more comparable with those of double parameter staining protocols (Figure 7.7).

7.2.5 Optimisation of Triple Staining Technique

Once a preliminary staining procedure was found, it could be refined. Addition of pepsin to the HCl dénaturation was tested to improve the access of the large R-phycoerythrin molecule to the DNA attached mouse monoclonal. Different pepsin concentrations were added to aliquots of the same cell sample for 20 minutes. DNA profiles were then examined, as demonstrated in Figure 7.6, for extent of damage and definition and the BrdUrd profiles analysed for greatest fluorescence (Table 7.2).

Table 7.2 Effect of different pepsin concentrations on BrdUrd staining Pepsin mg/ ml Total BrdUrd (%) Aneuploid BrdUrd Aneuploid BrdUrd (%) Mean Y Fluorescence 0 . 1 13.2 1138/6856 16.6 250 0.15 14.5 1254/6687 18.8 237 0 . 2 12.4 1145/6149 18.6 241 0.3 13.8 1184/6660 17.8 219 0.4 1 2 . 2 1025/6933 14.8 203 196

BrdUrd Profiles A NIT? Profiles o E c Cl 2 O ; . m m

!*■

rabbit biotin/R-phycoerythrin streptavidin

280 400 680 880 1800

D N A C o n ten t

rat biotin/R -phycoerythrin streptavidin

Cl <5 D N A C on ten t mouse R-phycoerythrin 200 400 600 800 1800 D N A C on ten t rabbit FITC 2 0 0 4 0 0 6 ^ ’ ' ê é é ’ ' 1000 D N A C o n te n t rabbit FITC

Figure 7.7 A single triple stained sample displaying the variation in

binding produced by different secondary antibody

There was little difference between digestion with 0.15 and 0.2m g/m l pepsin in 2M HCl, both yielding similar high percentages of aneuploid BrdUrd labelled cells, but 0.2m g/m l pepsin had slightly higher mean y (BrdUrd related) fluorescence with a better defined BrdUrd profile and was chosen as the optimum concentration of pepsin.

Varying lengths of time in 0.2m g/m l pepsin/2M HCl solution were then tested and compared to the standard preparation of 25 minutes in 2M HCl. The DNA profiles showed that at 10 minutes the DNA was not sufficiently denatured and at 30 minutes was being disrupted. At 25 minutes the BrdUrd staining was decreasing. The optimum time of 20 minutes was chosen as the percentage aneuploid figure was closest to that obtained with the standard technique (25 minutes in 2M HCl) used already in the laboratory (Table 7.3).

Table 7.3 Effect of different times in 0.2mg/ ml pepsin/ 2M HCl on BrdUrd staining Time minutes Total BrdUrd Aneuploid BrdUrd Aneuploid BrdUrd (%) % Aneuploid 1 0 542/2852 441/2232 19.8 78.3 15 563/3744 399/2137 18.7 57.1 2 0 589/3786 432/2329 18.6 61.5 25 481/3677 377/2364 15.9 64.3 30 521/3762 409/2549 16.1 67.8 25 HCl only - — 60.6

The hypoxia staining procedure was then analysed with the addition of pepsin to the dénaturation step for varying amounts of time. The DNA profiles were analysed using the SOBR model on Cellfit as described in 2.4.1.5. Hypoxia was defined by setting a region around the positive cells, by using a tumour not treated with NITP but taken through the staining procedure as a control sample. All the DNA profiles of the pepsin/HC l digests were better defined than digestion with HCl alone for 25 minutes as demonstrated previously with the BrdUrd staining (Figure 7.6).