Support & Resources

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Immunofluorescence Microscopy

Includes antibodies, bioconjugates, labeling kits, and CF® dyes for super-resolution applications
20 bright and photostable fluorescent CF® dye tyramides. Biotin & DNP conjugates also available
Universal antigen retrieval buffer for unmasking a wide variety of targets
Buffers specially formulated for blocking background from lipofuscin autofluorescence, charged dyes, and other sources
Wet-set and hard-set antifade mounting medium available with DAPI
Featuring permeabilization and fixation buffers, coverslip sealant, and pap pens

CF® Dye Antibodies and Other Conjugates

With superior brightness, photostability, and signal-to-noise, CF® dyes offer unrivaled performance for immunofluorescent applications.

Primary Antibodies

  • Growing collection of more than 1000 monoclonal antibodies: View flyer
  • Validated in IHC and other applications
  • Choose from 13 bright and photostable CF® dyes, R-PE, APC, PerCP, HRP, AP, or biotin
  • Available BSA-free, ready to use for Mix-n-Stain™ labeling
  • Affordable 100 uL sizes available

Browse primary antibodies, or search with Antibody Finder

Primary Antibodies – sizes and formats:

FormatConcentrationSize
CF® dye conjugates (13 colors)0.1 mg/mL100 uL or 500 uL
Biotin, HRP, or AP conjugates0.1 mg/mL100 uL or 500 uL
R-PE, APC, or Per-CP conjugates0.1 mg/mL250 uL
Purified, with BSA0.2 mg/mL100 uL or 500 uL
Purified, BSA-free (Mix-n-Stain™ Ready)1 mg/mL50 uL

Secondary & Anti-Tag Antibodies

  • Wide selection of secondary and anti-tag antibodies
  • Your choice of more than 20 CF® dye colors, R-PE, APC, HRP, AP or biotin
  • Highly cross-adsorbed, F(ab’)2 fragments, and isotype-specific options
  • Single label CF® dye conjugates for super-resolution STORM applications
  • Affordable 50 uL sizes available

Browse secondary antibodies, search with Antibody Finder, or see secondary antibody product listings with catalog numbers.

Secondary Antibodies – sizes and formats:

FormatConcentrationSize
CF® dye and biotin conjugates2 mg/mL50 uL, 500 uL, or 1 mg
HRP conjugates1 mg/mL100 uL, 1 mL, or 1 mg
AP conjugates1 mg/mL100 uL or 1 mg
R-PE conjugates0.5 mg/mL200 uL or 1 mL
APC conjugates0.5 mg/mL100 uL or 500 uL

HeLa cells stained with CellBrite™ Fix 555 (red), and CF®640R anti-mitochondrial marker clone 113-1 (cyan).
MCF7 cells stained with CF®555 anti-cyclin B1 clone CCNB1/1098 (red), CF®488A phalloidin (green), and DAPI.
HeLa cells stained with anti-Ki-67 (MKI67/2462) and CF®488A goat anti-mouse (green). Actin filaments are stained with CF®633 phalloidin (magenta).
Rat retina cryosection with mouse anti-neurofilament H and CF®568 goat anti-mouse (min x rat) (red), rabbit anti-GFAP and CF®488A goat anti-rabbit (green), followed by CF®640R anti-ZO1 (magenta), and DAPI.

CF® Dye Bioconjugates

We offer several CF® dye bioconjugates suitable for immunofluorescence microscopy workflows that are useful for visualizing cell surfaces, microtubules, apoptotic markers, and other cellular components.

  • Annexin V, nucleotide conjugates, and TUNEL assays for studying apoptosis
  • Glycoprotein probes and cell surface stains: ConA, WGA, and PNA lectins
  • Phalloidin conjugates for microtubule staining
  • Streptavidin and biotin conjugates
  • and more…

View our full selection of CF® dye bioconjugates here.

Antibody Labeling Kits

Labeling your own antibodies? Our Mix-n-Stain™ Antibody Labeling Kits offer convenient and stable labeling of IgG antibodies with 29 CF® dyes and other labels.

  • Label with your choice of 29 CF® dyes in only 30 minutes
  • Labeling Kits for FITC, Biotin, R-PE, APC and other labels also available
  • Stable covalent labeling with no purification required
  • Compatible with common antibody stabilizers.
  • Labeling kits designed for Nanobodies® (ie. camelid single variable or VHH domains) also available

Learn more about full selection of Mix-n-Stain™ Labeling Kits here.

Rat intestine cryosection stained with NucSpot® 470 (green) and CF®640R phalloidin (magenta).

 

HeLa cells treated with oleic acid complexed to BSA, then fixed and stained with LipidSpot™ 488 (green), CF®594 WGA (red), and Hoechst 33258 (blue).
Rat testis with mouse anti-tubulin and CF®488A goat anti-mouse (min x rat) (green), CF®555 Mix-n-Stain labeled mouse anti-ZO1 (tight junctions, red) and CF®640R phalloidin (cyan).
HeLa cells stained with mouse anti-tubulin and CF®543 goat anti-mouse (red), followed by CF®488A Mix-n-Stain™ labeled mouse anti-transferrin receptor (green).

CF® Dyes for Super-Resolution Applications

Several CF® dyes have demonstrated superior performance for super-resolution applications in recent publications. The superior brightness, photostability, and photochemical switching properties of our CF® dyes are ideal for 3-D SIM, multi-color 3-D STORM, and other super resolution and single molecule imaging techniques.

Learn More About CF® Dyes for Super-Resolution Imaging

See Super-Resolution References
Download the Super-Resolution Imaging Flyer
Find out more about CF® dyes

Comparison of conventional wide-field microscopy (left) with STORM (right) using CF® dye conjugates. Images courtesy of Sam Kenny and Professor Ke Xu, College of Chemistry, University of California, Berkeley.

Looking for organelle specific stains? View our Cellular Stains page for a complete list of products for staining nucleus, cytoplasm, mitochondria, membranes, lysosomes, and other cellular components.

Also view our Cellular Stains Comparison Guide for a quick reference on targets, applications, fixability, and more information on our cell stains.

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Tyramide Signal Amplification

Tyramide signal amplification (TSA) is a highly sensitive method enabling the detection of low-abundance targets in fluorescent immunocytochemistry (ICC), immunohistochemistry (IHC), and in situ hybridization (FISH) applications. TSA involves horseradish peroxidase (HRP)-catalyzed deposition of tyramide on and near a target protein or nucleic acid sequence in situ. We offer tyramide signal amplification kits with biotin and 6 of our bright and photostable CF® dyes as well as standalone dye and hapten labeled tyramides. We also offer optimized tyramide amplification buffers for enhancing sensitivity for TSA.

Tyramide Signal Amplification Kits:

  • Our kits provide all critical reagents for tyramide labeling
  • Choose your tyramide: biotin tyramide or one of six CF® dye tyramides
  • Choose your HRP conjugate: goat anti-mouse, goat anti-rabbit, or streptavidin
  • Kits also include Amplification Buffer and BSA for blocking

Tyramide Amplification Buffers:

  • Tyramide Amplification Buffer Plus has enhanced sensitivity for tyramide signal amplification. It has improved brightness, specificity, and sensitivity over our original buffer below.
  • Ready-to-Use Tyramide Amplification Buffer was our original buffer formulation, and has been replaced with the above buffer. The one advantage of this buffer is that there is no need to add hydrogen peroxide.

 

Tyramide Amplification Kit with HRP Goat Anti-Rabbit and CF® Dye or Biotin Tyramide
Illustration of the tyramide signal amplification system. A cell or tissue sample is labeled with primary and secondary antibody using conventional methods. The horseradish peroxidase, conjugated to the secondary antibody, catalyzes the conversion of labeled tyramide into a reactive radical. The tyramide radical then covalently binds to nearby tyrosine residues, providing high-density labeling

Standalone Dye and Hapten Labeled Tyramides

Tyramide label Ex/EmSizeCatalog no.
CF®350347/448 nm0.5 mg 92170
CF®405L395/545 nm0.5 mg92198
CF®405S 404/431 nm0.5 mg92197
CF®405M 408/452 nm0.5 mg96057
CF®430426/498 nm0.5 mg96053
CF®488A490/515 nm0.5 mg92171
FITC492/514 nm0.5 mg96018
CF®514 516/548 nm0.5 mg92199
CF®532 527/558 nm0.5 mg96066
CF®543 541/560 nm0.5 mg92172
CF®550R551/577 nm0.5 mg96077
CF®555 555/565 nm0.5 mg96021
Cyanine 555 (Cy®3) 555/565 nm0.5 mg96020
CF®568 562/583 nm0.5 mg92173
CF®583R586/609 nm0.5 mg96085
CF®594 593/614 nm0.5 mg92174
CF®620R 617/639 nm0.5 mg92194
CF®640R 642/662 nm0.5 mg92175
CF®647 650/665 nm0.5 mg96022
CF®660R 663/682 nm0.5 mg92195
CF®680R 680/701 nm0.5 mg92196
CF®750*755/777 nm0.5 mg96052
Biotin-XX N/A0.5 mg92176
DNP N/A0.5 mg96019

*CF®750 tyramide (catalog #96052) is offered as a custom product. Please contact Technical Support to inquire about placing an order.

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AntiFix™ Antigen Retrieval Buffer

AntiFix™ Universal Antigen Retrieval Buffer is designed for heat-induced epitope retrieval (HIER) of formalin-fixed, paraffin embedded (FFPE) tissues. The buffer has been formulated to provide excellent heat unmasking for a wide variety of targets at neutral pH, reducing the need for buffer optimization. It has been validated in immunofluorescence staining of FFPE tissue with our next-generation fluorescent CF® dyes using HRP-mediated tyramide signal amplification.

Features:

  • Excellent antigen retrieval for a wide variety of targets at neutral pH
  • Simplifies HIER optimization
  • Validated with fluorescence staining using tyramide amplification
  • Non-toxic and non-flammable

Staining of human FFPE tissues for a variety of targets after antigen retrieval using AntiFix™ Universal Amplification Buffer. Colon stained with monoclonal antibodies against ZO-1 (clone ZO1-1A12) or PanCK (clone C-11); tonsil stained with monoclonal antibodies against PD-1 (clone PDCD1/922) or CD8 (clone EP1150Y). Antibodies were detected using CF®488A tyramide signal amplification.

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TrueBlack® Background Reducers

TrueBlack® Lipofuscin Autofluorescence Quenchers

Lipofuscin autofluorescence in certain human or aged animal tissues fluoresce brightly in all channels, making immunofluorescence virtually impossible unless lipofuscin fluorescence is masked. Traditionally, Sudan Black B has been used to quench lipofuscin autofluorescence but also introduces non-specific red and far-red fluorescence,  limiting the use of fluorescent dyes in those wavelengths. Biotium’s line of TrueBlack® lipofuscin autofluorescence quenchers are a superior alternative to Sudan Black B to quench autofluorescence with much lower background (Figure 1). Learn more about our TrueBlack® Background Reducers.

New! TrueBlack® Plus: A unique lipofuscin quencher

TrueBlack® Plus is a next-generation lipofuscin quencher developed by Biotium chemists. This new quencher was designed to allow lipofuscin quenching in aqueous buffer with even lower background than the original TrueBlack® (Figure 1.). Quenching in PBS allows longer incubation times for thick samples without shrinkage, and is compatible with hydrophobic stains.

TrueBlack® IF Background Supressor

The TrueBlack® Background Suppressor System is a buffer system designed for optimal blocking of non-specific staining for immunofluorescence (IF). The buffers are designed to block background from both non-specific antibody binding as well as direct interaction of fluorescent dyes on antibodies with cells or tissue sections (Figure 2).

TrueBlack® IF Background Suppressor Features

  • Suppresses background from non-specific antibody binding and charged fluorescent dyes
  • More efficient than Image-iT® FX, block & permeabilize in just 10 minutes
  • Complete system for blocking, permeabilizing, and antibody dilution
  • Non-mammalian blocking agents, for broad secondary antibody compatibility
  • For immunofluorescence on cells or tissue sections

TrueBlack® and TrueBlack® Plus has lower background than Sudan Black B

Figure 1. Lipofuscin autofluorescence in methanol-fixed human cerebral cortex cryosection without quenching treatment appears as bright punctate spots that fluoresce in all channels, appearing pink the merged image. Traditional treatment with Sudan Black B introduces high levels of far-red background fluorescence. Treatment with original TrueBlack® Lipofuscin Autofluorescence Quencher eliminates lipofuscin autofluorescence, but still introduces low-level red/far-red fluorescence. TrueBlack® Plus can be used in PBS instead of 70% ethanol, and quenches lipofuscin autofluorescence with the lowest level of far-red background.

VIEW & PURCHASE

TrueBlack® Plus Lipofuscin Autofluorescence Quencher, 40X in DMSO
TrueBlack® Lipofuscin Autofluorescence Quencher

TrueBlack® IF Background Suppressor reduces background fluorescence due to charged dyes an non-specific antibody binding

TrueBlack IF Background Suppressor
Figure 2. TrueBlack® IF Background Suppressor reduces non-specific binding of antibodies conjugated to charged fluorescent dyes. Methanol-fixed HeLa cells were blocked for 10 minutes with fish gelatin blocking buffer or TrueBlack® IF Background Suppressor, then stained with no primary antibody or mouse anti-tubulin antibody, followed by goat anti-mouse secondary antibody conjugated to the indicated dyes. Antibodies were diluted in the same buffer used for blocking.

VIEW & PURCHASE

TrueBlack® IF Background Suppressor System (Permeabilizing) – 20 assays
TrueBlack® IF Background Suppressor System (Permeabilizing) – 200 assays

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EverBrite™ Mounting Medium

Biotium’s EverBrite™ line of mounting media are a revolutionary antifade mounting media optimally formulated for preserving fluorescence of our CF® dyes and other fluorochromes. EverBrite™ Mounting Medium is available in wet-set and hard-set formulations with and without DAPI. The EverBrite™  Hardset Mounting Medium hardens completely to form a permanent seal at room temperature in 24 hours. Drop-n-Stain EverBrite™ Mounting Medium provides the wet-set EverBrite™ formulation in a convenient dropper bottle for easy dispensing.

  • Antifade mounting medium optimally formulated to prevent photobleaching of fluorophores
  • Refractive index is well-matched to that of coverslip glass and immersion oil
  • Available in wet-set or hard-set formulations
  • Available with or without DAPI

Unlike VECTASHIELD® wetset, EverBrite™ does not quench the fluorescence of cyanine-based dyes. Methanol-fixed HeLa cells in a 96-well coversglass bottom dish were stained with mouse anti-tubulin antibody followed by Cy®5 goat anti-mouse secondary antibody, then placed in the indicated mounting medium and imaged by confocal microscopy using the same laser and gain settings. Other widely-used cyanine-based dyes include Cy®3, Alexa Fluor® 555, Alexa Fluor® 647, CF®555, CF®647, CF®660C, and CF®680.
Unlike VECTASHIELD® Hardset, EverBrite™ Hardset does not quench the fluorescence of cyanine-based dyes. Methanol-fixed HeLa cells on coverslips were stained with mouse anti-tubulin antibody followed by Cy®5 goat anti-mouse secondary antibody. Coverslips were mounted using the indicated medium, cured according to the manufacturer’s instructions, and then imaged by confocal microscopy. Other widely-used cyanine-based dyes include Cy®3, Alexa Fluor® 555, Alexa Fluor® 647, CF®555, CF®647, CF®660C, and CF®680.

EverBrite™ Mounting Media

ProductCounterstainCat. No.
EverBrite™ Mounting MediumNone23001
EverBrite™ Mounting Medium with DAPIDAPI23002
Drop-n-Stain EverBrite™ Mounting MediumNone23008
Drop-n-Stain EverBrite™ Mounting Medium with DAPIDAPI23009
EverBrite™ Hardset Mounting MediumNone23003
EverBrite™ Hardset Mounting Medium with DAPIDAPI23004

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Buffers and other accessories

CoverGrip™ Coverslip Sealant

CoverGrip™ Coverslip Sealant is the first product specifically designed for sealing the edges of wet-mounted coverslips for fluorescence microscopy. Unlike nail polish, CoverGrip™  contains no ingredients that can leach into aqueous mounting medium and affect specimen fluorescence.

  • Seals the edges of wet-mounted coverslips for fluorescence microscopy
  • Dries hard and clear
  • Less damaging to samples than nail polish
  • Won’t leach into aqueous mounting medium and affect fluorescence
  • Contains natural and environmentally friendly d-limonene instead of hazardous organic solvents

SuperHT Pap Pen

The SuperHT PAP Pen draws a water repellent circle around slide mounted tissue and prevents the waste of valuable reagents by keeping the liquid contained in a single droplet. Our SuperHT PAP Pens are available in regular size (~800 applications), or mini size (~400 applications).

  • Draw water repellent barriers around tissue samples on slides
  • Conserve precious antibodies and reagents
  • Barriers are insoluble to aqueous buffer, detergents, alcohols, and acetone
  • Barriers are stable at temperatures up to 120°C

 

Pap Pens can be used to create barriers around sections on slides, to conserve and/or segregate staining solutions.

See below a list of other products useful for immunofluorescence microscopy

ProductCatalog No.SizeFeatures
Fixation Buffer22015100 mL• Ready-to-use formaldehyde-based fixation buffer
Permeabilization Buffer22016100 mL• Ready-to-use permeabilization buffer for intracellular staining
Permeabilization and Blocking Buffer (5X)22017100 mL• Concentrated buffer for one step permabilization and blocking for intracellular immunofluorescence
• Goat serum-based blocking buffer
Flow Cytometry Fixation/Permeabilization Kit2300650 tests• Optimally formulated buffers for fixation/permabilization for intracellular staining for flow
10X Fish Gelatin Blocking Agent22010100 mL• Provides excellent blocking for immunofluorescence or western
• Add to buffer of your choice (PBS or TBS)
• Compatible with goat and sheep primaries, unlike BSA, milk, or goat serum
Fish Gelatin Powder220112 x 50 g• Gelatin from cold water fish skin for blocking for immunofluorescence or western
• Compatible with goat and sheep primaries, unlike BSA, milk, or goat serum
Bovine Serum Albumin, 30% Solution22014100 mL• Commonly used blocking agent and antibody or protein stabilizer
• 30% solution in water
• Made from IgG-free, protease-free Fraction V BSA
Bovine Serum Albumin Fraction V2201350 g• Commonly used blocking agent and antibody or protein stabilizer
• IgG-free, protease-free Fraction V BSA
Tween®-202200250 mL• Detergent commonly used for western blocking and washing
Mini Cell Scrapers22003Pack of 200• For harvesting cells or cell lysates from 96-, 48- and 24-well plates
• 0.5 cm (3/16″) wide and 6 cm (2 3/8″) long
• 20 packs of 10 scrapers per pack
• Polyethylene, disposable & sterile