Cells grown on coverslips (Kemtech #0340-0150) were fixed for 10 min in 2% (w/v) sucrose and 2% (v/v) paraformaldehyde at RT followed by PBS washes. Coverslips were blocked in 0.2% (w/v) fish gelatin and 0.5% (w/v) BSA in PBS. Cells were incubated with primary antibodies and after PBS washes, cells were incubated with appropriate Alexa fluor secondary antibodies followed by washes in PBS + 0.1% Triton. For IF-FISH, the cells were further fixed with 4% (w/v) paraformaldehyde for 10 mins, followed by hybridization with TelC-Cy3 (CCCTAA)3 PNA telomere probe (PNA Bio, F1002) in hybridization buffer (0.5 μg/ml tRNA, 1 mg/ml BSA, 0.06 × SSC, 70% formamide), denatured at 85 °C for 3 mins and then incubated at RT overnight in a humid chamber109 (link). After washing the coverslips, DNA was stained with DAPI (Vectashield # H1200), and digital images captured at 10 ms or 100 ms exposure using NIS-Elements BR (Nikon) with a Nikon Eclipse 80i microscope and an Andor CCD camera. For EdU detection, cells labeled with 20 µM EdU for 30 mins were fixed as above followed by IF-FISH. After PNA FISH, cells were fixed with 4% PFA for 4 mins. Fixed cells were blocked with 3% BSA for 30 mins at RT followed by EdU detection according to manufacturer’s protocol (Click-it EdU Alexa Fluor 488 imaging kit, Invitrogen #C10337).
F1002
Manufactured by PNA Bio
The F1002 is a high-performance laboratory centrifuge designed for general-purpose applications. It features a robust and durable construction, with a maximum speed of 6,000 RPM and a maximum RCF (Relative Centrifugal Force) of 4,500 x g. The centrifuge accommodates a variety of sample containers, including test tubes, microplates, and sealed bottles. Its user-friendly control panel allows for easy operation and precise speed and time settings.
Automatically generated - may contain errors
Lab products found in correlation
Nis elements br, by Nikon (1 mentions)
Click it edu alexa fluor 488 imaging kit, by Thermo Fisher Scientific (1 mentions)
Eclipse 80i microscope, by Nikon (1 mentions)
H 1200, by Vector Laboratories (1 mentions)
Demecolcine, by Merck Group (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Axioplan 2 fluorescent microscope, by Zeiss (1 mentions)
Tirf microscope, by Zeiss (1 mentions)
5 protocols using f1002
1
Immunofluorescence and FISH for Telomere Analysis
2
Fluorescence in situ Hybridization (FISH) and FISH-SCE Protocol
3
Metaphase Chromosome Analysis by FISH
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The metaphase chromosome preparation and FISH were performed as described (Waisertreiger et al., 2020 (link)). Briefly, day 3 stimulated primary B cells were arrested in metaphase by a 1 hr treatment with 0.1 μg/ml demecolcine (Sigma, D1925), treated with 0.075 M KCl, fixed in methanol:acetic acid (3:1), spread onto glass slides, and air-dried. FISH was performed on metaphase cells using IgH probe. Prior to hybridization, slides were briefly heated over an open flame, denaturing DNA for IgH detection. Slides were washed in 1× PBS at RT for 5 min, post-fixed in 1% formaldehyde at RT for 5 min, and washed in 1× PBS at RT for 5 min. Slides were dehydrated in ethanol (75, 85, and 100%) at RT for 2 min each and air-dried. Cells and probes were co-denatured at 75°C for 3 min and incubated overnight at 37°C in a humid chamber. Slides were washed post-hybridization in 0.4 × SSC/0.3% NP-40 at 72°C (2 min), then 2 × SSC/0.1% NP-40 at RT (2 min). Slides were probed with 0.25 μM telomere probe (PNA Bio, F1002) for 1 hr at RT. Slides were then washed in 1× PBST (1× PBS, 0.5% Triton-X-100) for 5 min at 37°C. After wash with PBS and dehydrated in ethanol (75, 85, and 100%), slides were counterstained with Vectashield mounting medium containing DAPI (Vector Laboratories Inc, H-1200) before microscopy.
4
Telomere FISH Analysis of Heart Tissue
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The telomere fluorescence in situ hybridization (FISH) assay was performed according to manufacturer's instructions (PNA Bio). Briefly, heart tissue sections (5 µm) were fixed with 4% paraformaldehyde, washed with PBS, and permeabilized with 0.2% Triton X‐100 in PBS, with subsequent washes. The slides and hybridization buffer (20 mmmol/L Tris and 60% formamide in distilled water) were prewarmed at 85°C for 5 minutes. The PNA probe (PNA Bio, F1002) was diluted in hybridization buffer (1:250) and added to the slide, covered with a plastic coverslip, and incubated at 85°C for 10 minutes, followed by 2 hours at room temperature in the dark. After removing the coverslip carefully, slides were washed (2× standard saline citrate, 0.1% Tween‐20) twice for 10 minutes at 60°C and 5 minutes at room temperature. The slides were then blocked with 1% BSA in PBS for 1 hour at room temperature and incubated overnight at 4°C with γH2AX antibody (1:500) in blocking buffer. The next day, slides were washed with PBS 3 times for 5 minutes and incubated with fluorescent secondary goat anti‐mouse Alexa Fluor 488 (1:500) for 1 hour at room temperature in the dark. Slides were then washed in PBS 3 times for 5 minutes, coverslips were mounted with Prolong Gold Antifade with DAPI, and images were taken with Zeiss Axioplan 2 fluorescent microscope.
5
Telomere Length Quantification by T-FISH
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Cells were grown in 7.5 mM BrdU and 2.5 mM BrdC for 14 h prior to treatment with colcemid at 10 mg/ml for 3 h prior to collection. The cells were then fixed, dropped over a wet slide at a 45° angle and then washed and dried overnight as described for telomere fluorescent in situ hybridization (T-FISH). Metaphase cell-containing slides were then rehydrated in PBS, stained with Hoechst 33258 and exposed to 365 nm UV light. Slides were then digested with ExoIII, washed in PBS and dehydrated in a cold ethanol series. Slides were then individually hybridized with a G- or C-strand-specific PNA probe conjugated to either Cy3 or AF488 (PNA Bio, F1008 and F1002). The slides were then washed, counterstained with DAPI and dehydrated in a cold ethanol series. Images were acquired on a Zeiss TIRF microscope and analyzed with FIJI software. Images were scored in a blind manner.
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