CHAPTER 5: In vivo and in vitro effects of injectable hormonal contraceptives on
6.3 Materials and Methods
6.3.2 Processing of vaginal biopsies
Cervicovaginal biopsies, snap frozen in liquid nitrogen, were embedded in optimum cutting temperature (OCT, Sakura, Torrance, CA, USA) medium in a cryomold to ensure optimum orientation and longitudinal sectioning of the epithelium and then transferred to -20°C. The frozen block was adhered to the cryosectioning chuck at -20°C to ensure the optimal temperature for sectioning, 12 µm sections were cut onto glass slides using the Tissue-Tek®
Cryo3® Plus Microtome/Cryostat (Sakura, Torrance, CA, USA), fixed with isopropanol and methanol, and stained with either (i) hematoxylin and eosin (H&E) (to distinguish the stratified squamous epithelium) and (ii) with fluorescently-labelled antibodies against HIV-1 target cells (Cyanine 5 labelled CD4 or DakoCytomation CD68 antibodies; to quantify HIV-1 target cell density within tissue).
For H&E staining, vaginal biopsy sections were transfered into isopropanol for two minutes, then into methanol for 2 minutes and then rinsed in running tap water for 1 minute. Sections were incubated in haematoxylin for 5 minutes and rinsed in running tap water for 5 minutes.
The slides were then incubated in eosin for 1 minute. Slides were rehydrated by dipping in absolute ethanol ~20 times. Slides were subsequently washed 3 times with tap water, remaining liquid suctioned and mounted in fluorescent mounting medium (DAKO S3023) and covered with a coverslip. The edges were sealed with clear nail varnish; dried in the dark for 15 minutes and stored in the slide box at 4°C until imaged using a Zeiss AxioScope light microscope interfaced with the AxioVision (4.8.2 version) under 10X magnification.
Immunohistochemical staining of CD4 and CD68 cells was performed to investigate target cell recruitment in vaginal mucosa, in the laboratory of Prof Thomas Hope at Northwestern
University, Chicago (S. Ngcapu, 2014 Fogarty training fellowship). The slides were warmed at room temperature for 5 to 10 minutes and tissue sections were outlined with a PAP pen (Sigma-Aldrich, MO,USA). Slides were fixed at room temperature by placing a freshly made solution of 3.7% formaldehyde in Pipes buffer onto the slide for 10 minutes. Slides were then washed 3 times with PBS and the remaining liquid was suctioned.
For CD4 staining, slides were incubated with 1:200 dilution of the conjugated primary antibody directed against CD4 (OKT4 mouse, Sigma-Aldrich, MO,USA) diluted in donkey serum blocking solution (10% normal donkey serum, 0.1% Triton X-100/0.01%NaN3 and 1X PBS) at room temperature for 60 minutes. Slides were washed 3 times with PBS, and the remaining liquid suctioned. Tissue sections were then incubated with hoechst DAPI (staining nuclear material; DAPI used at 1:25000 dilultion; Invitrogen, CA, USA) diluted in donkey serum blocking solution in the dark at room temperature for 10 minutes. The slides were washed 3 times with PBS, remaining liquid suctioned and the tissue sections were finally mounted in fluorescent mounting medium (DAKO S3023, CA, USA) using a 20 mm coverslip (Thermo Fisher Scientific, MA,USA). The edges of the coverslip were sealed with clear nail varnish; dried in the dark for 15 minutes and stored in slide box at 4°C until imaged.
For CD68 staining, slides were initially stained with a 1:200 dilution of unconjugated primary anti-CD68 (DakoCytomation clone EBM11, Dako, CA, USA; diluted in donkey serum blocking solution) at room temperature for 60 minutes. Slides were washed 3 times with PBS and the remaining liquid was suctioned. After washing, the slides were incubated with 1:500 dilution of the secondary antibody (donkey anti-mouse Rhodamine Red X in donkey serum
blocking solution) in the dark at room temperature for 25 minutes. Slides were then washed 3 times with PBS, remaining liquid suctioned. Finally, slides were then incubated with a 1:25000 dilution of hoechst DAPI (to stain nuclear material) diluted in donkey serum blocking solution in the dark at room temperature for 10 minutes. The slides were washed 3 times with PBS, remaining liquid suctioned and mounted in fluorescent mounting medium (DAKO S3023) and a coverslip applied. The edges of the coverslip were sealed with clear nail varnish; dried in the dark for 15 minutes and stored in slide box at 4°C until imaged.
Vaginal biopsy tissue sections, stained with H&E stains, were viewed on the Zeiss AxioScope Light Microscope interfaced with the AxioVision image analysis software programme (4.8.2 version, Zeiss, Oberkochen, Germany). Consecutive lengths (parallel lines) of mucosa were archived from each vaginal biopsy sample, with care taken to avoid oblique and transverse areas. Images were archived-1ed at 10x magnification and stored as .zvi files.
Slides were viewed in a blinded fashion by 3 independent assessors, with two assessors using the manual measurements (Sinaye Ngcapu and Dr Desh Archary, CAPRISA Mucosal Laboratory, Durban) and the third using automated measurements (Dr Ann Carias, Northwestern University, Chicago, USA; using specially created algorithms using IDL software, Exelis, VA, USA). Approximately 3 - 4 interactive length measurements of mucosal thickness were manually performed by drawing parallel lines from the epithelium- lamina propria border to the stratum basale (including any lamina propria papillae) and images generated were stored with MS.zvi extension. Information of evident microabrasions or tears in the mucosal epithelium was also noted for each sample. Using the automated system, algorithms in IDL software enabled drawing of two distinct lines, at the luminal surface and where cellular junctions were robust. The distance (thickness) was then calculated from each individual point from line A to the closest distance of another point on
line B, and vice versa. Approximately 2000-7000 interactive length measurements of mucosal thickness were performed per image analysed. Consecutive lengths of mucosal epithelium were archived from each sample with care taken to avoid oblique and transverse areas. This data was extrapolated onto an excel file for unblinding.
For immunofluorescent staining of HIV-1 target cells in vaginal biopsies, images were obtained by deconvolution microscopy on a DeltaVision RT system (Applied Precision, LLC, WA, USA) collected on a digital camera (CoolSNAP HQ; Photometrics, AZ, USA) using a 40x oil objective, and performed at the Hope Laboratory, Northwestern University, Chicago, USA. All images were archived at 40x magnification and stored as z-stacks. SoftWoRx analysis software (MT, USA) was used to measure the shortest distance of intra-epithelial target cells to the surface of tissue epithelium. The cell density was calculated using the following formula:
Nc/A; A = Ni x I1 x I2 x p2
Where Nc = number of cells, I1 = y pixel size, A = area surveyed, I2 = x pixel size, Ni = number of images, p2 = pixel size in μm squared