4.6. EVALUACIÓN TÉCNICA DE PETITORIOS MINEROS SUPERPUESTOS
4.7.3. EVALUACIÓN TÉCNICA DE PETITORIOS SUPERPUESTOS SOBRE
2.2.1 Preparation o f embryos
Time mated 11 day p.c. New Zealand white rabbits were killed using lethal intravenous overdose o f Euthatal (pentobarbitone 200mg/1.5kg body weight). Tq was taken as noon following an early morning mating. The uterus was opened under aseptic conditions in a lamina flow cabinet and decidual swellings removed and placed in prewarmed culture medium (BGJ). It proved impossible to separate the decidua from the yolk sac so embryos were freed from all surrounding membranes and rinsed in Hank’s balanced salt solution (BBSS).
2.2.2 Monensin treatment using grid culture
Embryos were placed on stainless steel grids in prewarmed, pregassed Dulbecco’s modification o f Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS; Gibco, Grand Island, New York), antibiotics (lOOIU/ml penicillin V; lOOpg/ml streptomycin and 2.5|rg/ml amphotericin B) and monensin (5|j,M, Sigma), and cultured at 37°C for either 6,12 or 24 hours in an humidified atmosphere o f 5% CO^ in air.
2.2.3 Monensin treatment using roller culture
Embryos were placed in prewarmed and pregassed 30ml tubes (Sterilin) 3 embryos per tube, containing either 5ml DMEM with 25% i.c. rabbit serum (Appendix I) or 5ml 100% i.c. rabbit serum, supplemented with antibiotics and monensin (5pM). Tubes were regassed for 2 minutes with 5%C02/air and incubated in a roller culture apparatus (30rpm) at 37°C for either 12 or 24 hours.
2.2.4 Monensin treatment using *in vivo* culture
This additional experiment was designed to test the effectiveness o f vivo* monensin treatment. A single 29 day p.c. time mated New Zealand white rabbit was dosed by oral gavage with monensin (lOmg/kg) in a gelatin capsule 24 hours prior to killing and embryo collection. Embryos were removed under aseptic conditions and the distal femoral growth plates dissected (a positive control sanq)le known to synthesize MMPs, especially stromelysin: Brown et a l, 1989) and either jfrozen immediately as described below or prepared for in vitro grid culture. Samples for further monensin treatment were placed in warmed dissecting medium (BGJ), cleaned and cut in half along the mid- sagittal plane. The cut surface was placed face down on a stainless steel grid and cultured as for grid culture above. Subsequently sanq)les were mounted and frozen.
2.2.5 Freezing o f samples
Following culture, whole embryos were prefixed in freshly prepared 4% paraformaldehyde in PBS (phosphate buffered saline; Appendix I) at 4°C for 1 hour and cryoprotected in 30% sucrose at 4°C for 1 hour. AU samples were mounted in Tissue- tek, incubated for 1 hour at 4°C to aUow penetration o f mountant and frozen slowly in a bath o f thawing isopentane suspended in nitrogen vapour. Sandies were stored at -70°C until required for immunolocahzation.
2.2.6 Antisera
Specific polyclonal antibodies to rabbit coUagenase (S-anti-CL), gelatinase (S-anti-GL; which recognizes both gelatinase A and B), stromelysin (S-anti-SL) and TIMP-1 (S-anti- TIMP-1) were raised in sheep (a gift from. Dr. Ros Hembry, SRL, Cambridge). The characterisation o f these antisera, including species specificity. Western blots, inhibition
curves and immunoabsorption experiments with purified antigen, has been reported in detaü elsewhere (Hembry et al., 1986; Gfavrilovic et al., 1987; Murphy et al., 1986; Murphy et a l, 1989b). Pooled normal sheep serum (NSS) was used as a control. The second antibody for indirect immunolocahzation was either a fluorescein isothiocyanate labelled monovalent Fab’ preparation from an antiserum raised in a pig (pig-FITC) as previously described (Hembry et a l, 1985) and diluted 1:300 in PBS, or fluorescein isothiocyanate conjugated anti-sheep IgG (whole molecule) raised in a donkey (Sigma; donkey-FITC), and diluted 1:200 in 5% normal donkey serum (Sigma) in PBS.
2.2.7 Immunolocalization
Serial frozen tissue sections (4-6 frm) were cut and mounted on APES coated shdes (Appendix I). Sections were air dried for 5 minutes. Unfixed tissue was fixed for 30 minutes and prefixed embryos fixed for a further 10 minutes in freshly prepared 4% paraformaldehyde in PBS, washed in PBS and permeabilized (0.1% Triton X-100 in PBS) for 5 minutes to facihtate IgG penetration o f cell membranes. They were then incubated with either NSS, S-anti-CL, S-anti-GL, S-anti-SL or S-anti-TIMP (IgG; 50 p, g/ml in PBS, 30 minutes) in a humidified atmosphere. After repeated washing in PBS, sections were incubated with second antibody (pig-FITC or donkey-FITC, 30 minutes), washed in PBS and counterstained with a nuclear stain (methyl green, I mg/ml, 2 minutes) which fluoresces red when visualized with a wide band FITC or rhodamine filter. Sections were mounted with Citifluor (Glycerol/PBS, City University, London, England), covershpped and sealed with glyceel (Gurr, BDH). All sections were then examined within 24 hours by fluorescence microscopy on either a Zeiss photomicroscope
in
with epifluorescence and standard wide band FITC filters or a Zeiss standard WL microscope equipped with rhodamine and standard narrow band FITC filter sets.2.2 8 Con focal microscopy
Further embryos were prepared for whole mount examination in a confocal microscope as follows. Briefly, whole embryos were flxed in freshly prepared 4% paraformaldehyde for 1 hour, rinsed well in PBS, permeabilized in Tiiton-X 100 over night, rinsed well in PBS and incubated with primary antibodies either S-anti-CL, S-anti-GL, S-anti-SL or S- anti-TIMP for 48 hours. Following prolonged washing in PBS (24 hours) embryos were incubated with second antibody (donkey-FITC) for 48 hours. Following further prolonged washing in PBS embryos were briefly counterstained in propidium iodide, washed in PBS, mounted in Citifluor and examined using a confocal microscope.
2.3 Results
2.3.1 in vitro embryo culture
Monensin-treated embryos, either grid or roller cultured, retained adequate macroscopic anatomy during the culture period. However, normal development during culture was not determined for this pilot study. All embryos were observed to have beating hearts at the end o f the culture period albeit at a reduced rate.
2.3.2 in vivo embryo culture
Oral dosage o f the pregnant rabbit with monensin was easily accomphshed appeared to have no detrimental effect on the animal. Growth plate cartilage prepared from embryos taken from this animal showed normal hitological morphology (data not shown).
2.3.3 Immunoloclaization o f MMPs and TIMP-1 in embryos
Sections o f in vitro monensin-treated embryos prepared for immunofluorescence microscopy and incubated with antisera to coUagenase, gelatinase, stromelysin and TIMP-1 foUowed by FITC-labeUed second antibody showed red counterstained nuclei and only faint non-specific background fluorescent green staining (data not shown). Control sections from monensin-treated embryos incubated with NSS IgG foUowed by FITC-labeUed second antibody showed only red counterstain nuclei (data not shown).
2.3.4 Immunoloclaization o f MMPs and TIMP-1 in embryonic growth plate cartilage
Trial and error selection o f prefixation, cryoprotection and slow freezing produced frozen sections with adequate morphology for tissue identification (data not shown). Sections from in vivo monesin-treated growth plate sarq)les incubated with antisera for coUagenase, gelatinase, stromelysin and TIMP-1 foUowed by FITC-labeUed second antibody showed only occasional faint intraceUular staining for MMPs and TIMP-1 and red counterstained nuclei (data not shown). The level of staining observed was similar to that detected in non-monensin treated tissues. Sarrples o f in vivo monesin-treated growth plate subsequently monensin-treated using in vitro grid culture and incubated with antisera for MMPs and TIMP-1 showed bright intraceUular fluorescence and red counterstained nuclei (data not shown). The bright green staining seen in these sections represents antigen synthesized and accumulated during subsequent in vitro culture, indicating penetration and action of monensin.
2.3.5 Confocal microscopy
Whole mount monensin-treated embryos prepared for confocal microscopy and incubated with antisera to coUagenase, gelatinase, stromelysin and TIMP-1 foUowed by FITC-labeUed second antibody showed counterstained nuclei and bright non-specific background staining. Control monesin-treated satrq)les incubated with NSS IgG foUowed by FITC-labeUed second antibody showed only counterstained nuclei. Using optical sectioning, no patterns o f bright fiuorescent staining could be determined at any level.