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Hcx pl fluotar 63 1.25 na oil immersion objective

Manufactured by Leica camera
Sourced in Germany

The HCX PL Fluotar 63x/1.25 NA oil immersion objective is a high-performance microscope objective designed for fluorescence imaging. It has a magnification of 63x and a numerical aperture of 1.25, which provides excellent spatial resolution and light-gathering capability. The objective is optimized for use with oil immersion, which enhances its optical performance.

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5 protocols using hcx pl fluotar 63 1.25 na oil immersion objective

1

Microscopic Visualization of Pre-40S Subunits

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Cells were visualized using a DM6000B microscope (Leica, Germany) equipped with a HCX PL Fluotar 63×/1.25 NA oil immersion objective (Leica). Images were acquired with a fitted digital camera (ORCA-ER; Hamamatsu Photonics, Japan) and Openlab software (Perkin–Elmer, USA).
Localization of pre-40S subunits was monitored employing the uS5-GFP reporter construct as previously described (Faza et al., 2012 (link); Altvater et al., 2014 (link)). Co-localization of Slx9-GFP and Slx9-1-GFP with Gar1-mCherry was done as previously described (Faza et al., 2012 (link)).
The heterokaryon assay was adapted and modified from (Belaya et al., 2006 (link); Altvater et al., 2012 (link)). Briefly, equal amounts of cells expressing Enp1-GFP, Gar1-GFP, or Slx9-GFP were mated with kar1-1 cells expressing Nup82-mCherry and concentrated onto 0.45-µM nitrocellulose filter. Mixtures were placed on YPD plates containing 50 µM cycloheximide. After 1 hr incubation at 30°C, cells were analyzed by fluorescence microscopy.
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2

Localization of 20S pre-rRNA and Pre-40S subunit export

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Localization of 20S pre-rRNA was analyzed using a Cy3-labeled oligonucleotide probe (5′-Cy3-ATG CTC TTG CCA AAA CAA AAA AAT CCA TTT TCA AAA TTA TTA AAT TTC TT-3′) that is complementary to the 5’ portion of ITS1 as previously described (Faza et al., 2012 (link)).
Pre-40S subunit export, monitored by localization of uS5-GFP and localization of GFP-tagged Fap7 and uS11 was performed as previously described (Altvater et al., 2014 (link); Faza et al., 2012 (link); Pertschy et al., 2007 (link); Milkereit et al., 2001 (link)). Cells were visualized using a DM6000B microscope (Leica, Germany) equipped with a HCX PL Fluotar 63×/1.25 NA oil immersion objective (Leica, Germany). Images were acquired with a fitted digital camera (ORCA-ER; Hamamatsu Photonics, Japan) and Openlab software (Perkin-Elmer, USA). All Cell-biological studies were performed at least on three different occasions and in triplicates; >99% of cells showed the reported phenotypes in a sample size of >1000 cells.
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3

Ribosome Biogenesis Visualization

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Cells were grown to mid-logarithmic phase and fixed with 4% formaldehyde in 0.1 M potassium phosphate buffer. Cells were then converted to spheroplasts using 0.1 M potassium phosphate buffer containing 1.2 M sorbitol and 500 μg of zymolase. Spheroplasts were washed in 2× SSC buffer and incubated overnight at 37 °C with Cy3-labeled oligonucleotide probe (5′-Cy3-ATG CTC TTG CCA AAA CAA AAA AAT CCA TTT TCA AAA TTA TTA AAT TTC TT-3′) that is complementary to the 5′ portion of ITS159 (link). DNA was stained with 0.5 μg/ml DAPI. Early biogenesis defect of small ribosomal subunit was determined by localization of uS5-GFP expressed from centromeric plasmid pRS316-RPS2-GFP59 (link),60 (link). Cells were visualized using a DMI6000 microscope (Leica, Germany) equipped with a HCX PL Fluotar 63×/1.25 NA oil immersion objective (Leica, Germany). Images were acquired with a fitted digital camera (ORCA-ER; Hamamatsu Photonics, Japan) and Openlab software (Perkin-Elmer, USA).
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4

Localization of 20S pre-rRNA and Pre-40S Subunit Export

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Localization of 20S pre-rRNA was analyzed using a Cy3-labeled oligonucleotide probe (5′-Cy3-ATG CTC TTG CCA AAA CAA AAA AAT CCA TTT TCA AAA TTA TTA AAT TTC TT-3′) that is complementary to the 5′ portion of ITS1 as previously described (Faza et al., 2012 (link)).
Pre-40S subunit export, monitored by localization of uS5-GFP and localization of GFP-eS26 was performed as previously described (Faza et al., 2012 (link); Altvater et al., 2014 (link)). Indirect immunofluorescence using affinity-purified polyclonal antibodies against the TAP-tag (1:1000; Thermo Scientific; Rockford, IL, USA) and staining of the nuclear and mitochondrial DNA with DAPI was performed as described previously (Schlenstedt et al., 1997 (link); Solsbacher et al., 1998 (link)).
Cells were visualized using DM6000B microscope (Leica, Germany) equipped with HCX PL Fluotar 63 × /1.25 NA oil immersion objective (Leica, Solms, Germany). Images were acquired with a fitted digital camera (ORCA-ER; Hamamatsu Photonics, Hamamatsu, SZK, Japan) and Openlab software (Perkin–Elmer, Waltham, MA, USA).
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5

Localization of 20S pre-rRNA and Pre-40S Subunit Export

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Localization of 20S pre-rRNA was analyzed using a Cy3-labeled oligonucleotide probe (5′-Cy3-ATG CTC TTG CCA AAA CAA AAA AAT CCA TTT TCA AAA TTA TTA AAT TTC TT-3′) that is complementary to the 5′ portion of ITS1.40 (link)Pre-40S subunit export, monitored by localization of uS5-GFP and localization of GFP-eS26.40 (link),41 (link)Cells were visualized using a DM6000B microscope (Leica, Germany) equipped with a HCX PL Fluotar ×63/1.25 NA oil immersion objective (Leica, Solms Germany). Images were acquired with a fitted digital camera (ORCA-ER; Hamamatsu Photonics, Hamamatsu, SZK, Japan) and Openlab software (Perkin-Elmer, Waltham, MA, USA).
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