In brief, cells were fixed with 4% paraformaldehyde for 10 mins, then 100% cold methanol for 10 mins and followed by 70% ethanol for 10 mins for rehydration, cells were then incubated with 1M Tris pH8.0 for 5 mins before incubated with 1ng/μl Cy3-Oligo-dT (30) for rat beta-actin in hybridization buffer (2xSSC with 1mg/mL Yeast tRNA, 0.005% BSA, 10% Dextran sulfate, 25% formamide) in 37oC overnight. For single molecule FISH, rat ActB-Quasar570 smFISH probes were designed using the Stellaris Probe Designer software (Biosearch Technologies), smFISH was performed according to manufacturer’s instruction using Stellaris buffers (Biosearch Technologies SMF-HB1-10, SMF-WA1-60 and SMF-WB1-20). After hybridization, cells were washed with 4xSSC and 2xSSC once each, and incubated with primary antibody in 2xSSC+ 0.1% triton-X-100 at 4oC overnight, then washed 3 times with 2xSSC and incubated with fluorescence labeled secondary antibody in 2xSSC+ 0.1% triton-X-100 at room temperature for 1hr. Coverslips were mounted and imaged with Zeiss Airyscan.
Smf wa1 60
Manufactured by Biosearch Technologies
Sourced in United States
The SMF-WA1-60 is a laboratory equipment product. It is designed to perform specific functions within a laboratory setting. The core function of this product is to provide a controlled environment or specialized capability to support various laboratory operations, but no further details about its intended use or capabilities can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Smf hb1 10, by Biosearch Technologies (15 mentions)
Smf wb1 20, by Biosearch Technologies (11 mentions)
Stellaris probe designer, by Biosearch Technologies (4 mentions)
Lsm 880, by Zeiss (4 mentions)
Airyscan, by Zeiss (2 mentions)
Smf wb1 20, by Vector Laboratories (2 mentions)
Tamra dye, by Biosearch Technologies (1 mentions)
Stellaris fish probe designer, by Biosearch Technologies (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Vectashield hardset antifade mounting medium with dapi, by Vector Laboratories (1 mentions)
Dfc365fx, by Leica camera (1 mentions)
Stellaris rna fish protocol, by Biosearch Technologies (1 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
Stellaris rna fish probe, by Biosearch Technologies (1 mentions)
Evos fl auto imaging system, by Thermo Fisher Scientific (1 mentions)
Cal fluor red 590, by Biosearch Technologies (1 mentions)
X software, by FlowJo (1 mentions)
Xist rna fish, by Biosearch Technologies (1 mentions)
Tcs sp8 confocal microscope, by Leica camera (1 mentions)
Quasar 570, by Biosearch Technologies (1 mentions)
19 protocols using smf wa1 60
1
Multimodal Single-Cell Transcriptomics Protocol
2
Single-cell mRNA and Protein Imaging
3
Quantitative RNA FISH for Murine CCL2
4
Visualizing Nascent Histone RNA in Adherent Cells
5
RNA FISH Staining of Paraffin-Embedded Tissue
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U1 in situ probes were designed and ordered in the Stellaris Probe Designer (Biosearch Technologies) (Table 3 ). Tissue sections were de‐paraffinized and stained following Biosearch Technologies Stellaris RNA FISH protocol for formalin‐fixed paraffin‐embedded tissue. Briefly, tissue sections were washed in Wash Buffer A (Biosearch Technologies, SMF‐WA1‐60), before adding 200 μl hybridization buffer (Biosearch Technologies, SMF‐HB1‐10) containing the U1 probe and covering the tissue with a glass coverslip. The slides were then incubated overnight in a humid box at 37°C. Slides were then immersed in Wash Buffer A in the dark at 37°C for 30 min, allowing the coverslips to float off. Slides were then washed for 5 min with Wash Buffer B (Biosearch Technologies, SMF‐WB1‐20), before sections were mounted with VECTASHIELD® Hardset™ Antifade Mounting Medium with DAPI (Vector Laboratories, H‐1500) for nuclear staining. All imaging was done on a DFC365FX (Leica) at 63× magnifications.
6
RNA-FISH Visualization of SERPINE1 Expression
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RNA-FISH followed the manufacturer’s instructions (Biosearch Technologies, Inc., Petaluma, CA). The custom SERPINE1 RNA-FISH probes consisted of 47 fluorescently Quasar 570-labeled probes complementary to different regions of the human SERPINE1 mRNA transcript. Each probe was 20 nucleotides in length (Supplementary Table S11 ). The tissue sections were fixed in 4% formaldehyde for 10 min at room temperature. They were washed twice in PBS and permeabilized in 70% ethanol for 1 hour at room temperature. The sections were washed with buffer A [10% (v/v) formamide in 1× wash buffer A; Biosearch Technologies, Hoddesdon, UK, catalog no. SMF-WA1-60] and incubated with hybridization buffer (Biosearch Technologies, catalog no. SMF-HB1-10) containing 125 nM RNA-FISH probes in the dark within a humidity chamber at 37 °C overnight. The sections were washed with buffer A, stained with DAPI, and washed with buffer B (Biosearch Technologies, catalog no. SMF-WB1-20). Coverslips were mounted on glass slides using Prolong Diamond Antifade Mountant (Invitrogen). Images were acquired as already described.
7
Hmrhl Transcript Detection by RNA-FISH
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RNA-FISH was carried out by following the protocol from Biosearch Technologies (https://www.biosearchtech.com/products/rna-fish ). A set of Stellaris RNA FISH Probes labeled with Cy5 dye specific to Hmrhl targeted transcript was purchased from Biosearch Technologies. Briefly, 5 × 106 cells were washed with PBS, followed by fixation in 1 ml fixation buffer (37% formaldehyde in PBS) for 10 min at room temperature. Cells were then centrifuged and washed thrice with PBS before permeabilizing with 70% ethanol for at least 1 h at 2–8°C. Cell pellet was washed once with washed buffer A (Biosearch Technologies, Cat #SMF-WA1-60) and then proceeded for hybridization with 100 ul hybridization buffer (Biosearch Technologies, Cat #SMFHB1-10) containing Hmrhl probe (working concentration 125 nM) for overnight at 37°C. Next day, cells were washed once with wash buffer A and resuspended in the same to be incubated for 30 min at 37°C in the dark. After incubation, cells were centrifuged and resuspended in wash buffer A containing DAPI stain and incubated further for 30 min at 37°C in the dark. Next, cells were washed with wash buffer B (Biosearch Technologies, Cat #SMF-WB1-20) and resuspend in a small drop (∼30 μl) of Vectashield Mounting Medium. Subsequently, 10 ul of cell suspension was mounted on a slide for microscopic observation.
8
In Situ Detection of miR-200b-3p in Virus-Infected Cells
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FISH probes labeled with the Quasar 570 fluorophore for detecting miR-200b-3p were purchased from GenePharma (Shanghai, China). 293T cells infected with IAV (MOI = 0.01, 24 h) or VSV (MOI = 0.01, 16 h) were fixed for 10 min with 4% paraformaldehyde for in situ hybridization analysis and then washed with PBS three times. Cells were permeabilized with 0.2% Triton X-100 for 10 min and washed briefly. Cells were then incubated with wash buffer A (SMF-WA1-60, Biosearch Technologies, Petaluma, CA, USA) for 5 min. Wash buffer A was removed, and the hybridization buffer (SMFHB1-10, Biosearch Technologies) containing FISH Probes (final concentration, 12.5 nM) were added and incubated for 16 h at 37°C in the dark. The hybridization buffer was then removed, and cells were incubated with wash buffer A for 5 min at 37°C. Cells were then stained with DAPI for 10 min at room temperature. Cells images were obtained with an EVOS FL Auto imaging system (Thermo Fisher Scientific) after being washed with PBS three times.
9
Visualizing Pcl mRNA in Drosophila Ovaries
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We ordered custom Stellaris RNA FISH oligonucleotide probes directed against Pcl mRNA conjugated to CALFluor RED 590 from LGC Biosearch Technologies and performed in situ hybridization based on the company’s recommendations. Briefly, we washed fixed ovaries 2 × 20 min. in Wash Buffer A (LGC Biosearch Technologies #SMF-WA1-60) + 10% formamide (WAF) then incubated ovaries for at least 2 hr in hybridization buffer (LGC Biosearch Technologies #SMF-HB1-10) + 10% formamide (HBF) at 37°C. We then incubated ovaries in HBF + 50 nM probe overnight, before washing 1x with HBF, 3x WAF at 37°, and 2x Wash Buffer B (LGC Biosearch Technologies #SMF-WB1-20) at room temperature. We mounted ovaries in 50% glycerol and imaged on a Leica SP8 scanning confocal.
10
Fluorescence In Situ Hybridization for LncKdm2b RNA
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FISH probes were designed using the Stellaris Probe Designer (Biosearch Technologies) (See Table S4 for primers used in FISH probes). A total of 38 probes with 20 nucleotides in length were used (Tsingke Biotech). Probes were biotinylated using terminal transferase (NEB, M0315S) with Bio-N6-ddATP (ENZO, ENZ-42809) as substrates. To detect LncKdm2b RNA, adherently cultured primary NPCs derived from E13.5 mouse cortex were rinsed in PBS and then fixed with 3.7% formaldehyde in PBS at room temperature for 10 min. Cells were permeabilized with 70% ethanol at 4 °C overnight. Cells were treated with RNase A (100 µg/mL) or with PBS (in the control group) at 37 °C for 1 h. After washing in Wash Buffer A (Biosearch Technologies, SMF-WA1-60) for 5 min, cells were incubated with DNA probes in hybridization buffer (Biosearch Technologies, SMF-HB1-10) at 37 °C overnight. After hybridization, cells were incubated with Alexa Fluor 555 conjugated streptavidin (1:1500 diluted in 1% BSA in PBS) at RT for 1 h. Cells were washed twice with Wash Buffer A at 37 °C for 30 min followed by nuclear counterstaining with DAPI. For colocalization studies, cells were co-stained with mouse anti-hnRNPAB (Santa Cruz Biotechnology).
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