The SenTraGor™ Immunohistochemistry Protocol has been standardized using 4.5 μm tissue sections. If you would like to use thicker tissue sections you need to optimize the protocol and increas incubation times for the SenTraGor™ compound and the antibodies.
For co-IHC staining SenTraGor™ reagent must be applied at the end of the process after the antigen retrieval step (see co-IHC SenTraGor™ Protocol).
So, for staining with 3 different antibodies you must follow the same tactic and apply SenTraGor™ reagent at the end of the staining protocol.
!Notice!: before the application of SenTraGor™ reagent you must incubate tissue with sequential solutions of 50% EthOH and 70% EthOH (see steps 3.6 and 3.7 in IHC SenTraGor™ Protocol).
In the publication of the discovery of SenTraGor™ compound, Aging Cell (2017), Supporting Information S2, there are information on the exact reagents used for the validation and development of SenTraGor™ reagent. The primary anti-biotin antibody used in all the applications is from Abcam, product code ab201341. For IHC and ICC, the inventors are using the Ultravision Quanto Detection System HRP DAB kit, ThermoFisher Scientific, catalog # TL-125-QHD, which includes its own secondary antibodies. For IF and Flow Cytometry analysis, the inventors use the secondary fluorescently labeled antibody from ThermoFisher Scientific, catalog # A-11004. We have not used so far the HRP substrates from VectorLab, but someone can use SenTraGor™ with the reagents that have been standardized in the lab.
As indicated in the publication on Aging Cell (2017), in Supporting Information S2, the anti-biotin antibody used for validation of SenTraGor™ reagent in all its applications is from Abcam, product code ab201341. The secondary fluorescently labeled antibody that was used for the SenTraGor™ Flow Cytometry Protocol is from ThermoFisher Scientific, catalog # A-11004.
In theory you could use a secondary fluorescent antibody streptavidin conjugated. But from our experience the direct method doesn't work. You need the primary anti-biotin antibody to enhance the signal of SenTraGor™ reagent and then proceed with a secondary antibody against your primary antibody.
We stain routinely megakaryocytes in bone marrow samples with SenTraGor™ reagent. Actually such samples are provided as positive controls with the reagent.
To use the anti-biotin antibody in tissue samples with endogenous biotin you will need to block the endogenous biotin. Before applying SenTraGor™ reagent you will have to use a Streptavidin/Biotin blocking kit. Follow the instructions on SenTraGor™ Immunohistochemistry Protocol, Note 4.7.
The cellular systems that can be used as positive controls for staining with SenTraGor™ reagent are cells undegoing replicative senescence or cells exhibiting Stress Induced Senescence.
Animal tissues that can be used as positive controls for staining with SenTraGor™ reagent are the animal models and human clinical cases with established senescence. For example: K-RAS induced mouse lung adenomas, irradiated human laryngeal lesions, histological sections from bone marrow that include diffusely distributed normal megakaryocytes that undergo a senescence process during their differentiation.
You can also advise the Datasheet of the reagent.
The method for using SenTraGor™ reagent in powdered-crushed tissue or solutions is under development and soon it will be available under SenTraGor™ Protocols.
Yes. SenTraGor™ reagent is a chemical compound and not an antibody. It is not species specific. It is chemically attached to lipofuscin inside senescent cells from any origine.
You need to check one of the following steps to make sure of technical correctness.
Yes, SenTraGor™ reagent can be applied to frozen sections.
If preservation of morphology is not crucial you can proceed with the Immunocytochemistry SenTraGor™ Protocol from step 3.1
If preservation of morphology is essential, material can be fixed in 1% (w/v) formaldehyde/PBS for 5 min, then 3x washings (approx. for 1 min each) in PBS and stained by continuing the Immunohistochemistry SenTraGor™ Protocol from step 3.3
Theoreticaly you can use directly HRP-streptavidin. But from our experience the results are more reproducible, specific and consistent when you use the anti-biotin antibody. The signal of the SenTraGor™ reagent is enhanced with the primary anti-biotin antibody and secondary antibody against the primary antibody.
No. SenTraGor™ reagent can only be applied to fixed biological material. The reagent must be diluted in ethanol which means that any treatment of the cells with the compound will result in their death and fixation.
SenTraGor™ reagent is a chemical compound, a biotynilated Sudan Black B analogue, for the detection of lipofuscin inside senescent cells. It is not an antibody, but you need an anti-biotin antibody to enhance the signal of SenTraGor™ staining.