Digoxigenin (DIG)-labeled and fluorescein-labeled riboprobes were prepared and used for ISH as described previously (Atkinson-Leadbeater et al., 2010 (link); Yang et al., 2018 (link)). Briefly, riboprobes were transcribed in vitro using SP6 or T7 polymerase (Roche), DIG-labeled or fluorescein-labeled ribonucleotides (Roche), and linearized plasmid templates pBSK-xfgfr1, pBSK-xBek-ec, pBSK-xfgfr3, pBSK-xfgfr4, pCRII-xsema3A, and pCMV-SPORT6-slit1 (Golub et al., 2000 (link); Atkinson-Leadbeater et al., 2009 (link), 2010 (link), 2014 (link)). The specificity of all riboprobes was assessed through sense controls. For color development of wholemount ISH, tissues were incubated with anti-DIG alkaline phosphatase-conjugated Fab fragments (Roche catalog #11 093 274 910; RRID: AB_2313640 ) and stained with BM Purple (Roche; Sive et al., 2000 ). For double fluorescent ISH (dFISH) on sectioned tissue, samples were incubated with anti-DIG peroxidase-conjugated (Roche catalog #11 207 733 910; RRID: AB_514500 ) or anti-fluorescein peroxidase-conjugated (Roche catalog #11 426 346 910; RRID AB_840257 ) Fab fragments and stained with the TSA Plus Fluorescein Evaluation kit (PerkinElmer) and the TSA Cyanine 3 System (PerkinElmer).
Tsa plus fluorescein evaluation kit
Manufactured by PerkinElmer
Sourced in United States
The TSA Plus fluorescein evaluation kit is a laboratory equipment product designed for the detection and measurement of fluorescein. It provides a method for evaluating the performance of fluorescein-based assays or applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Bm purple, by Roche (1 mentions)
Sp6 or t7 polymerase, by Roche (1 mentions)
Alkaline phosphatase conjugated anti dig fab fragments, by Roche (1 mentions)
Tsa cyanine 3 system, by PerkinElmer (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Advanced Cell Diagnostics (1 mentions)
Rnascope protease 3, by Advanced Cell Diagnostics (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Fv1200 confocal microscope, by Olympus (1 mentions)
Rnascope multiplex fluorescent v2 kit, by Advanced Cell Diagnostics (1 mentions)
α sarcomeric actin, by Merck Group (1 mentions)
Anti digoxigenin antibody, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
6 protocols using tsa plus fluorescein evaluation kit
1
Riboprobe Synthesis and In Situ Hybridization
2
VISTA Expression Evaluation in Gynecological Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IHC staining was performed as previously described27 (link),28 (link) using anti-VISTA polyclonal antibody (Atlas Antibodies HPA007968, lot: A89595) or monoclonal antibodies (AMAb91252, lot: 03077; AMAb91253, lot: 03078; Atlas Antibodies, Bromma, Sweden). Two independent gynaecological pathologists blinded to the clinical data examined the stained sections. Staining scores were calculated based on the degree of staining (−, 0; +, 1; ++, 2; +++, 3) and percentage of area stained. Staining scores were calculated as percentage ×0 (degree −) + percentage ×1 (degree +) + percentage ×2 (degree ++) + percentage ×3 (degree +++).
Immunofluorescence of VISTA on CD8+ T cells involved double staining with antibodies to VISTA (AMAb91252, lot: 03077; Atlas Antibodies) and CD8 (clone: C8/144B; DAKO, Carpinteria, CA, USA) using TSA Plus fluorescein evaluation kit (PerkinElmer, Waltham, MA, USA).
Immunofluorescence of VISTA on CD8+ T cells involved double staining with antibodies to VISTA (AMAb91252, lot: 03077; Atlas Antibodies) and CD8 (clone: C8/144B; DAKO, Carpinteria, CA, USA) using TSA Plus fluorescein evaluation kit (PerkinElmer, Waltham, MA, USA).
3
Whole-mount In Situ Hybridization Protocol
4
Combinatorial Fluorescence In Situ Hybridization and Immunohistochemistry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemistry was performed as described previously [44 (link)]. To examine the co-staining of GFP or DsRed with npsn WISH, embryos were first incubated with an npsn antisense probe as described previously, except that the signal was expanded using a TSA Plus fluorescein evaluation kit (NEL741E001KT; PerkinElmer, Waltham, MA, USA) or TSA Plus cyanine 3 evaluation kit (NEL744E001KT; PerkinElmer). For antibody staining, embryos were first stained with goat anti-GFP or rabbit anti-DsRed antibody (1 : 200) at 4°C overnight and subsequently visualized by Alexa Fluor 488 donkey anti-goat (1 : 400) at 4°C overnight or Alexa Fluor 555 donkey anti-rabbit (1 : 400) 4°C overnight, respectively.
5
RNA-FISH Analysis of Fixed Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues were fixed and sectioned as previously described in methods for immunohistochemistry and immunofluorescence. RNA-FISH was performed using the RNAscope Multiplex Fluorescent v2 kit (323100; Advanced Cell Diagnostics, Newark, CA), following the protocol for Fixed Frozen Tissue. Briefly, tissue was pretreated with RNAscope Hydrogen Peroxide (322335; Advanced Cell Diagnostics), target retrieval was performed for 5 minutes, and tissue was treated with RNAscope Protease III (322337; Advanced Cell Diagnostics). Then, specified probes were hybridized using the RNAscope HybEZ II Oven (321710/321720; Advanced Cell Diagnostics). Probes were then amplified and the HRP signal was developed using TSA Fluorescein Plus Evaluation Kit (NEL741E001KT; Perkin Elmer, Waltham, MA). Finally, slides were counterstained with DAPI (323108; Advanced Cell Diagnostics) and mounted with ProLong Gold antifade reagent (P10144; Life Technologies Corporation, Eugene, OR). Imaging was performed on an Olympus FV1200 Confocal Microscope, in which multiple 60× images were taken per sample, of which 2–3 samples were used for each timepoint (day 7 vs day 70) in each treatment (sham vs SBR). Images were subsequently analyzed using unbiased computational quantification and representative images were chosen.
6
miR-34a Detection in Cardiac Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MiR-34a was detected by FISH according to protocol previously described [54 (link)] and modified as follow. Tissues sections were digested by 0.5μg/ml proteinase-K for 10′ at RT. miRCURY LNA detection probe hsa-miR-34a, 5′-DIG and 3′-DIG labeled and control probe LNA U6 snRNA, 5′-DIG labeled (Exiqon) were used at 50 nM and 10 nM respectively and denatured as suggested by manufacturer's instructions. To detect signal Anti-Digoxigenin antibody (Abcam) and TSA Fluorescein Plus evaluation kit (Perkin Elmer) were used. rCPCs were identified by c-kit (Santa Cruz Biotechnology), myocytes were labeled with α-sarcomeric actin (Sigma-Aldrich) and nuclei were stained with DAPI (Sigma-Aldrich). Samples were analyzed with a Zeiss LSM700 confocal microscope.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!