Axioscop 2 microscope

Fresh lung tumor tissues were obtained from patients who were undergoing surgical resection of the primary tumor. All human tissue samples were obtained and analyzed in accordance with procedures approved by the institutional review board of the University of California, San Francisco (IRB H8714–22 942–01). The tissue microarray sections were immunostained as previously described [55 (link)]. The following scoring system was used: −, no stain; +, weak staining (10% or above stained cellularity considered as positive); ++, moderate staining (30% or above stained cellularity considered as positive); +++, strong staining (50% or above stained cellularity considered as positive). All scoring was done under low power objective lens (20 ×) with a Zeiss Axioscop 2 microscope (Carl Zeiss Inc, Germany). Images were taken under 20 × or 40 × objective lens.

Fresh mesothelioma and adjacent normal pleural tissues were obtained from patients with mesothelioma who were undergoing surgical resection of the primary tumour. Primary human mesothelioma samples from 60 patients were fixed in formalin and embedded in paraffin in 4‐μm tissue microarray sections. In seven of these patients, a small amount of normal pleura tissue had been obtained simultaneously to serve as control. All human tissue samples were obtained and analysed in accordance with procedures approved by the institutional review board of the University of California, San Francisco (IRB H8714–22 942–01). The sections were immunostained as previously described [21, 22]. The following scoring system was used: −, no stain; +, weak staining (≥10% and <30% stained cellularity considered as positive); ++, moderate staining (≥30% and <50% stained cellularity considered as positive); +++, strong staining (50% or above stained cellularity considered as positive). All scoring was carried out under a low power objective lens (20×) with a Zeiss Axioscop 2 microscope (Carl Zeiss Inc, Oberkochen, Germany) by two independent, blinded researchers. Images were taken under 10× or 20× objective lens.

SW480 cells were cultured by the overlay method of 3D culture on solidified Matrigel (BD Biosciences, San Jose, CA) [30 (link)]. Briefly, cells were grown to ~ 80% confluency in a monolayer. A single cell suspension was prepared in RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin (100 units/ml), streptomycin (100 μg/ml) (Life Technologies) and 2% Matrigel and seeded onto 80 μl Matrigel in an 8-well chamber slide (BD Biosciences) at 2000 cells/well. All cultures were then maintained at 37°C in a 5% CO₂ humidified incubator for up to 10 d. Cell morphology was examined every alternate day and the size of resulting spheroids was measured on day 10 and volume was calculated by formula V = (4/3)πr³. Image analysis was done using Zeiss Axioscop 2 microscope (Carl Zeiss, Inc., Germany) and Carl Zeiss Axiovision Rel 4.5 software.

Fresh lung tumor tissues were obtained from patients who were undergoing surgical resection of a primary tumor. All human tissue samples were obtained and analyzed in accordance with procedures approved by the institutional review board of the University of California, San Francisco (IRB H8714–22 942–01). The tissue microarray sections were immunostained as previously described [48 (link)]. The following scoring system was used: −, no stain; +, weak staining (10% or above stained cellularity considered as positive); ++, moderate staining (30% or above stained cellularity considered as positive); +++, strong staining (50% or above stained cellularity considered as positive). All scoring was done under low power objective lens (10×) with a Zeiss Axioscop 2 microscope (Carl Zeiss Inc, Germany). Images were taken under 10× or 20× objective lens.

For teratoma formation, we used SCID hairless outbred mice (Crl:SHO-Prkdc^scidHr^hr) of SPF status and BALB/c-nu. Experiments with SCID mice were performed in the Center for Genetic Resources of Laboratory Animals (RFMEFI61914X0005) at ICG SB RAS; BALB/c-nu mice were kept in the Vivarium for conventional animals at ICG SB RAS. Teratomas were produced using standard protocol [45 ]. Between 3 and 17.7 × 10⁶ ES cells on passages 7-23 and 1.5 to 7 × 10⁶ iPS cells on passages 5-14 were injected subcutaneously into immunodeficient mice. Teratomas generated by ES or iPS cells were dissected after 3-12 weeks and were fixed in Bouin solution. Paraffin sections were prepared according to standard protocol and were stained with histological dyes Picro-Mallory trichromica (04-021822), Masson trichromica (04-011802), P.T.A.H.-hematoxyline (04-060802), Luxol fast blue Krever Barrera (04-200812), Azan trichromica (04-001802), Picrofuchsin Van Gizon (04-030802) (Bio-Optica Milano S.P.A., Italy) and with hematoxylin-eosin. Images were analyzed on Carl Zeiss Axioscop 2+ microscope with AxioCam HRc CCD-camera. Digital images were taken using AxioVision software in collective Microscopic Center of ICG SB RAS.
All animal studies were undertaken with prior approval from Interinstitutional Bioethical Committee of ICG SB RAS.

Tissue was fixed in 9 parts ethanol:1 part acetic acid for 2 h, then washed in 90% ethanol twice. Gynoecia were hand-dissected in ethanol and then moved into Hoyer's solution (70% ethanol, 5% gum arabic, 4% glycerol) for clearing and mounting on slides for visualization. Slides were examined with an Axioscop2 microscope (Zeiss) with Nomarski optics. Ovule counts were made from stages 11–14 gynoecia fixed on slides. Analysis of carpel bending and splitting was done under dissecting scope. Gynoecia were rated from 1 to 4 independently for bending as well as splitting. A severity score was given based on the following scoring system: 1, no defect; 2, mild defect; 3, moderate defect; 4, severe defect. All gynoecia were scored by the same individual, at the same time without knowledge of the genotype. All photos were captured with Q Capture software on a 5.0 RTV digital camera (Q Imaging, Surrey, BC, Canada). Data analysis was conducted in JMP Pro 10 (SAS Institute Incorporated, Cary, NC, USA) using multiple pair-wise comparison of the means with a Tukey-Kramer HSD test at an alpha of 0.05 or with a Student's T-test.