Stains

Antibodies

DiOC6(3)

Invitrogen 273

DiOC6(3) is a cell-permeant, green-fluorescent, lipophilic dye that is selective for the **mitochondria:: of live cells, when used at low concentrations. At higher concentrations, the dye may be used to stain other internal membranes, such as the endoplasmic reticulum.

ER Tracker

ER Tracker Red

(BODIPY™ TR Glibenclamide), for live-cell imaging Invitrogen/Life/ ThermoFisher: E34250

Ex = 587 nm Em = 615 nm

MW = 915.23

100 ug lyophilized.

To make stock solution:

  • Add 110 uL DMSO to make a 1mM solution
  • Make 1 uL aliquots

To use

Working concentration: ~1 uM.

  • Add 1 mL HBSS to 1 uL aliquot to make 1uM solution. (Use buffered saline with Ca++ and Mg++)

  • treat cells with warm 1 uM solution 15 - 30 min

  • rinse staining solution out

  • put non CO2 medium in dish

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Fluo-4 AM

Fluo-4 stock
Invitrogen F14217 500 µL

Calcium indicator (fluoresces when bound to Calcium ions)

Ex 494 nm; Em 516 nm

1mM in DMSO
Protect from light
Store in dissicator.
20 µL aliquots. Each makes 10 mL of 2 µM solution

Fluo-4 staining solution
2 µM Fluo-4 + 0.02% pluronic in HBS

Incubate 15 - 60 min at 20 - 37C. Wash before viewing.

20 % w/v Pluoronic

plus HBS to final volume

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Mitotracker

Invitrogen M7512

https://www.thermofisher.com/order/catalog/product/M7513?SID=srch-srp-M7513

Ex = 579 nm; Em = 599

50 ug per tube.

MW = 531

Add 100 uL DMSO to 50 ug in tube. Makes ~ 1 mM stock solution.

Use at 25 - 100 nM. (1 uL per 10 mL for 100 nM)

Dilute in medium. Treat with pre-warmed stain solution for 15 - 45 minutes. Replace staining solution with fresh (warm) medium.

To fix: use 3.7% formaldehyde (in medium); 37C for 15 minutes (??)

NBD Ceramide

Invitrogen N22651

fluorescent marker for Golgi in live cells

Ex: 466nm Em: 536nm

Follow instructions attached: Add 150 µL sterile H2O to the 5 mg in the bottle. (Makes 0.5mM (=500 µM) in BSA)

Aliquot 10 µL, and freeze

Working concentration is 5 µM

Add 990 µL HBS to 10 µL in tube

OR 99µL 10x HBS + 891 µL water

Aliquot ~120 µL per group (makes 8 aliquots)

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PS-Speck

Fluorescent Beads

Molecular Probes P7220

Code Color Ex (nm) Em (nm) filter cube label
A blue 360 440 UV
B green 505 515 B
C orange 540 560 LP (?) G?
D deep red 633 660 probably not
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Phalloidin

Alexa Fluor 568 Phalloidin (Also have green phalloidin in Histology stains)

Fisher or Invitrogen A 12380

Binds to F-Actin

Ex/Em 578/600

Stock Solution: Add 1.5 mL MeOH to vial (~6.6µM)

Aliquots

  • 5 µL stock per 0.5 mL tube
  • Label: Add 200 µL PBS (or PBS 1% BSA), use 50µL per coverslip

Fix cells with formaldehyde

Procedure:

  • 50 uL phalloidin on parafilm in humid chamber
  • coverslip cell side down onto drop
  • 15 - 20 minutes at room temp
  • rinse repeatedly in PBS-Tween-azide
  • blot the corner on a kimwipe
  • Mount on a drop of mounting medium on a slide
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Syto RNASelect

SYTO RNASelect Green Fluorescent Cell Stain (Invitrogen S32703)

$210 from Invitrogen

Fluoresces green when bound to RNA.

excite: 490 nm, emit: 530 nm

Can be used in live or fixed cells. Fix in methanol, NOT formaldehyde!

Don't use in conjunction with red-orange dyes.

Stock:

100µL 5 mM in DMSO. Store at -20C, dessicated, dark.

To thaw: warm to RT, spin down.

Should be stable for >= 1 year.

Make labeling solution

Make 5µM intermediate stock:

2 µL 5mM stock + 1998 µL medium or PBS

Make 20 100 µL aliquots in 1.5 mL tubes

Label: RNASelect - 100 µL - 5 µM intermediate stock in PBS; Add 900 µL to make labeling solution

Make 500 nM labeling solution in medium or PBS

100 µL 5 µM intermediate + 900 µL medium

Protocol

Live cells

  • Cells on coverslip
  • Warm 500 nM labeling solution to 37C
  • Incubate at 37C 20 min
  • Rinse twice in PBS or medium
  • Add warm medium, let cells rest 5 min
  • Fix in chilled methanol 10 min at -20C
  • Several washes in PBS

Fixed cells

  • Remove coverslip from medium
  • Fix in chilled methanol 10 min at -20C
  • Remove methanol, let slip sit in PBS 5 min
  • Apply labeling solution 20 min RT
  • Wash 5 min in PBS
  • Mount coverslip
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TMRE

Tetramethrylrhodamine ethyl ester perchlorate

Red fluophore

Biochemika 87917

Soluble in DMSO, alcohols.

Stock is 50 mM in DMSO

λex 540 nm; λem 595 nm in DMSO

cell-permeant, cationic, red-orange fluorescent dye that is readily sequestered by active mitochondria.

Potential-sensitive probe for measuring membrane potential changes in mitochondria

Transferrin

Alexa-Fluor 488 transferrin (Invitrogen T13342, MW = ~80KD) stock is 5 mg/ml (=62.5 µM).

Final concentration = 1 µM.

Aliquot 2.5 µL.

Add 150 µL Fe-HBS-BSA to make 153 µL 1 µM

Store in freezer

Transferrin is a monomeric serum glycoprotein (~80,000 daltons) that binds up to two Fe3+ atoms for delivery to vertebrate cells through receptor-mediated endocytosis.

Once iron-carrying transferrin proteins are inside endosomes, the acidic environment favors dissociation of iron from the transferrin–receptor complex. Following the release of iron, the apotransferrin is recycled to the plasma membrane, where it is released from its receptor to scavenge more iron.

Fluorescent transferrin conjugates can therefore be used with fluorescent LDL to distinguish the lysosomally directed and recycling endosomal pathways.

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