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XAF Black™ Ultra Lipofuscin Autofluorescence Blocker
Product key features
- Highly water soluble
- Optimized to block the autofluorescence from lipofuscin
- Extremely effective for blocking other autofluorescence as well
- More effective than other quenching products for histochemical staining
Product description
XAF Black™ Ultra Lipofuscin Autofluorescence Blocker is a recent upgrade of our outstanding XAF Black™ Lipofuscin Autofluorescence Blocker. It has significantly enhanced quenching capability and greatly improved water solubility. It is specifically formulated to reduce autofluorescence from lipofuscin. XAF Black™ Ultra Lipofuscin Autofluorescence Blocker can be much more effectively used to block the autofluorescence of lipofuscin than other similar products on the market. It can also be used to reduce other background fluorescence, as well as autofluorescence from other sources such as collagen, elastin, and red blood cells. It is a material that can accumulate in aged human and animal tissues. Lipofuscin is a heterogeneous amalgam mainly composed of oxidized proteins (30 to 70%) and lipids such as triglycerides, free fatty acids, cholesterol, and lipoproteins (20 to 50%). Carbohydrates make a small contribution that proportionally may increase with age (4 to 7%). It is generally considered that the protein content has a significant contribution from mitochondria, e.g., ATP synthase subunit residues in congenital ceroid lipofuscinoses. Lipofuscin accumulates in the lysosomes of many cell types with age and/or in patients with severe malnutrition and cancer cachexia. Due to its broad excitation and emission spectra (400 to 700 nm) the presence of lipofuscin complicates the fluorescence imaging of tissues employing exogenous detection fluorophores. The spectrum of lipofuscin overlaps with those of almost all the commonly used detection fluorophores, making it difficult or even impossible to distinguish between specific labelling and autofluorescence caused by lipofuscin. It often hampers fluorescence-based techniques if not properly addressed and corrected for. Autofluorescence is the natural emission of biological substances such as NAD(P)H in liver and vitamin A in hepatic stellate cells. Several other endogenous fluorophores are also known to cause autofluorescence in many tissues.
Example protocol
AT A GLANCE
Perform fixation, deparaffinization, and antigen retrieval of tissue sections as per your standard protocol.
Add XAF Black™ Ultra Lipofuscin Autofluorescence Blocker working solution to the sample.
Incubate the sample at room temperature for 5 to 10 minutes.
Apply the mounting medium and examine the sample using fluorescence microscopy.
Thaw the XAF Black™ Ultra Lipofuscin Autofluorescence Blocker DMSO solution at room temperature. If any precipitates are observed, gently heat the vial at 37°C for 5 to 10 minutes before preparing the working solution.
PREPARATION OF WORKING SOLUTION
To prepare the XAF Black™ Ultra Lipofuscin Autofluorescence Blocker working solution (1X), dilute 25 μL of the XAF Black™ Ultra Lipofuscin Autofluorescence Blocker 40X DMSO stock solution in 1 mL of PBS. Mix thoroughly.
Note: Protect the working solution from light by covering it with foil or storing it in a dark location
Note: For optimal results, use this solution within a few hours of preparation.
Note: Prepare 100 to 200 µL of XAF Black™ Ultra Lipofuscin Autofluorescence Blocker working solution (1X) for each tissue section that will be treated.
SAMPLE EXPERIMENTAL PROTOCOL
Perform fixation, deparaffinization, and/or antigen retrieval of the tissue sections according to your standard protocols.
Wash slides with PBS.
Remove the PBS, then place the tissues in a humidified slide chamber.
Add 100 to 200 µL of XAF Black™ Ultra Lipofuscin Autofluorescence Blocker working solution (1X) to each tissue sample, ensuring the solution fully covers the samples.
Incubate the slides at room temperature for 5 to 10 minutes.
Note: The incubation time may vary based on your specific application and can be adjusted for optimal results.
Wash the slides two times with PBS.
Perform immunofluorescence staining with antibodies according to your recommended protocol.
Note: Avoid using buffers containing detergents during blocking, antibody incubation, or washing steps. If detergents are necessary, use them according to the post-treatment protocol.
Cover the slides using any aqueous-based fluorescence anti-fade mounting medium, such as FluoroQuest™ PLUS Antifade Mounting Medium (AAT Cat# 20008).
Note: Avoid using organic-based mounting mediums, as they are incompatible with XAF Black™ Ultra Lipofuscin Autofluorescence Blocker.
Perform fixation, deparaffinization, and/or antigen retrieval of the tissue sections according to your standard protocols.
Perform immunofluorescence staining with antibodies according to your recommended protocol.
Note: Avoid using buffers containing detergents during blocking, antibody incubation, or washing steps. If detergents are necessary, use them according to the post-treatment protocol.
Wash the slides with PBS.
Remove the PBS, then place the tissues in a humidified slide chamber.
Add 100 to 200 µL of XAF Black™ Ultra Lipofuscin Autofluorescence Blocker working solution (1X) to each tissue sample, ensuring the solution fully covers the samples.
Incubate the slides at room temperature for 5 to 10 minutes.
Note: The incubation time may vary based on your specific application and can be adjusted for optimal results.
Wash the slides two times with PBS.
Cover the slides using any aqueous-based fluorescence anti-fade mounting medium, such as FluoroQuest™ PLUS Antifade Mounting Medium (AAT Cat# 20008).
Note: Avoid using organic-based mounting mediums, as they are incompatible with XAF Black™ Ultra Lipofuscin Autofluorescence Blocker.
References
Authors: Rami, Mohammad and Ahmadi Hekmatikar, Amirhossein and Rahdar, Samaneh and Marashi, Sayed Shafa and Daud, D Maryama Awang
Journal: Scientific reports (2024): 7133
Authors: Veloso Pereira, Beatriz Maria and Zeng, Yuting and Maggiore, Joseph C and Schweickart, Robert Allen and Eng, Diana G and Kaverina, Natalya and McKinzie, Sierra R and Chang, Anthony and Loretz, Carol J and Thieme, Karina and Hukriede, Neil A and Pippin, Jeffrey W and Wessely, Oliver and Shankland, Stuart J
Journal: American journal of physiology. Renal physiology (2024): F120-F134
Authors: Bragato, Cinzia and Persico, Andrea and Ferreres, Guillem and Tzanov, Tzanko and Mantecca, Paride
Journal: International journal of nanomedicine (2024): 7731-7750
Authors: Bacha, Samar and Arous, Fatma and Chouikh, Emna and Jaouani, Atef and Gtari, Maher and Charradi, Khaled and Attia, Hamadi and Ghorbel, Dorra
Journal: 3 Biotech (2023): 415
Authors: Zweyer, Margit and Ohlendieck, Kay and Swandulla, Dieter
Journal: Methods in molecular biology (Clifton, N.J.) (2023): 465-480
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