Stains

Stains kdorfman Thu, 08/13/2015 - 20:47

Acridine Orange

Acridine Orange kdorfman Fri, 07/27/2018 - 18:06

Sigma A8097

10 mg/mL solution in water

MW = 308.81

Ex: 486 - 492
Em: 516 - 532

From the Sigma web page:

Application

Acridine Orange hydrochloride solution has been used to study autophagic cell death. It has also been used for the staining of chromosomes.

Biochem/physiol Actions

Acridine Orange is a metachromatic dye which can stain DNA, RNA and acid glycosaminoglycans. At low concentration it intercalates into DNA and precipitates RNA. However, at high concentration it denatures and precipitates both RNA and DNA. Acridine Orange is also used to analyze autophagy. It goes into acidic organelles in a pH-dependent manner. At neutral pH, acridine orange gives a green fluorescence and in acidic conditions, it accumulates in the acidic organelle giving a bright red fluorescence.

From use in MBoMS: Use at ~20µM

  • 1.2 µL added to 2 mL in dish

  • incubate 15 min at 37

  • change medium

  • incubate 15 min at 37

  • Replace medium with PBS

  • observe with

    • B excite - G emission: dsDNA
    • G excite - R emission: RNA, ssDNA

Got good results initially, but within minutes, cells started to ball up and pull off the substrate. Will try new PBS first, then a concentration gradient of acridine orange.

Old PBS produced fast shrinking and balling up. Newer PBS was less bad. Now try with non-CO2 medium. Does it interfere with fluorescence?

No cell shrinkage in non-CO2 medium. At short exposure times, there is a background glow, but picking the right exposure takes care of that.

This is time sensitive. The red emission (which should be RNA & ssDNA) gradually fades, or overlies the green emmision (which should be ds DNA)

See attached images, taken in order 1, 2, 3. #3 is at about 15 minutes. 1 & 2 are 100x, 3 is 200x.

Click-iT® Plus EdU Imaging Kits

Click-iT® Plus EdU Imaging Kits kdorfman Mon, 08/12/2019 - 18:55

Materials required but not in kit:

Stock Solutions

Component conc dilute with
A (EdU) 10 mM 2 mL DMSO (C) or buffer
B (Alexa Fluor® picolyl azide)
C (DMSO)
D (reaction buffer) 10X
E Cu protectant
F (buffer additive)
G (Hoechst® 33342)

Per tube, need 200uL

To make mL Click-iT cocktail
add: uL D (reaction buffer)
add: uL Copper protectant (E)
add: uL Alexa Fluor (B)
and: uL F (buffer additive)

Buffer Additive

Buffer Additive kdorfman Thu, 03/26/2020 - 14:18

To make a 10X stock solution of the Click-iT® EdU buffer additive (Component F): Add 2 mL deionized water to the vial, then mix until fully dissolved. After use, store any remaining stock solution at ≤–20˚C.

When stored as directed, this stock solution is stable for up to 1 year. If the solution develops a brown color, it has degraded and should be discarded.

EdU

EdU kdorfman Wed, 09/11/2019 - 17:00

33mM (10X) Stock solution

100 mg of EdU powder (purchased from Carbosynth) https://www.carbosynth.com/carbosynth/website.nsf/(w-productdisplay)/D1…

is dissolved in

1.2 mL DMSO, then brought to 12 mL with H2O

Original recipe says: 10.8mL 0.5x E2 medium and

1.2ml DMSO

Rolf says the final concentration of DMSO should be more like 0.1% (after dilution of the 10X EdU to 1X in fish water)

Says to mix with water and warm gently (to thaw, use 60C for 15 minutes)

So try 33 mM EdU in 1% DMSO.

To make 20 mL 10X EdU in 1% DMSO, mix

  • 166 mg EdU
  • 0.2 mL DMSO (heat gently by putting it in a beaker of heated water)
  • bring to final volume with distilled water

Reaction Buffer

Reaction Buffer kdorfman Thu, 03/26/2020 - 14:13

Prepare a working solution of 1X Click-iT® EdU reaction buffer (Component D):

Transfer the solution (4 mL) in the Component D bottle to 36 mL of deionized water.

To make smaller amounts of 1X Click-iT® EdU reaction buffer,

  • dilute volumes from the Component D bottle 1:10 with deionized water.

  • After use, store any remaining 1X solution at 2–8˚C.

  • When stored as directed, this 1X solution is stable for 6 months.

DAPI in glycerol for fish

DAPI in glycerol for fish kdorfman Thu, 02/24/2022 - 19:36

1 mg/mL DAPI stock solution from Rolf

1 uL per 10 mL 25% glycerol

DNA gel stains

DNA gel stains kdorfman Mon, 08/27/2018 - 20:57

3-color LD + SYBR Safe

3-color LD + SYBR Safe kdorfman Mon, 10/15/2018 - 17:38

The orange runs fast; suitable for small DNA fragments

To make 10 mL from home made LD:

  • 6 mL orange G (Fisher AAJ60562AC)
  • 1 mL home made LD with bromophenol blue and xylene cyanacol
  • 6 uL SYBR safe
  • 1 mL glycerol
  • 0.1 mL 1 M Tris pH 7.6
  • water to 10 mL

EtBr

EtBr kdorfman Mon, 08/27/2018 - 21:01

Ethidium bromide

1 uL / 100 mL gel (or DNA sample, if adding it to the loading dye) (=1/100,000)

10 mg/mL
10 mL
17-ETBC1001 Krackeler $37.12

Home-made

Home-made kdorfman Mon, 08/27/2018 - 21:13

6X Home-made loading dye:

  • 30% glycerol
  • 0.3% Bromphenol blue
  • 0.3% xylene cyanol

plus SYBR-Safe at 0.5 uL/1mL

1 uL loading dye stains 5 uL of DNA sample.

SYBR safe works at 1/10,000

need 5/10,000 uL for 5 uL of sample

so need 5/10,000 uL SYBR safe for each uL of loading dye.

= 5/10 uL for each mL

NOTE Fisher Tritrack loading dye (FERR1161) composition:

  • 0.03% bromophenol blue
  • 0.03% xylene cyanol
  • 0.15% orange g
  • 60% glycerol
  • 10 mM Tris, pH 7.6
  • 60 mM EDTA

One tenth as much dye, twice as much glycerol.

SYBR Safe

SYBR Safe kdorfman Mon, 08/27/2018 - 21:04

SYBR safe DNA gel stain

Fisher S33102

400 uL $72.62

Use 1 uL/10 mL gel or DNA sample (=1/10,000)

Safe-Green

Safe-Green kdorfman Mon, 08/27/2018 - 21:13

Safe-Green

(DNA stain + loading dye)

ABM G108-G
1 mL $65.00 ($30 + shipping)

Use at 1:5

Smart-glow

Smart-glow kdorfman Mon, 08/27/2018 - 21:07

Smart Glow loading dye

Krackeler 26510-E4500-LD
1mL $77.22

DNA stain + loading dye.

Add directly to DNA sample

Use at 1:5

DiOC6(3)

DiOC6(3) kdorfman Fri, 05/11/2018 - 16:44

Invitrogen D273

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 kdorfman Thu, 04/05/2018 - 15:34

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

Fluo-4 AM

Fluo-4 AM kdorfman Thu, 04/26/2018 - 18:04

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

Lysotracker

Lysotracker kdorfman Thu, 10/24/2019 - 20:17

Lysotracker (Invitrogen L-7528 - 20 x 50 µL)

50 - 75 nM
stock = 1 mM in DMSO
aliquot 10 µL in 2 mL tubes, so they can make 2 mL medium
30 min - 2 hours incubation warm.

Mitotracker

Mitotracker kdorfman Wed, 02/21/2018 - 18:59

Mitotracker GREEN

Mitotracker GREEN kdorfman Thu, 10/24/2019 - 20:34

Thermo fisher M7514

Excitation 490 nm : Emission 516 nm

MW: 674

50 ug in vial.

Add 74 uL DMSO to make 1 mM stock.

working concentration =~ 25 - 200 nM

1 uL 1mM stock into 10 mL medium makes 100 nM treatment solution

Mitotracker RED

Mitotracker RED kdorfman Thu, 10/24/2019 - 20:34

Invitrogen M7512

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

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

NBD Ceramide kdorfman Wed, 04/25/2018 - 14:32

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)

To use:

  • incubate in cold

  • Minimal rinse! Very gently! Cold cells will fall off.

Nuc Blue

Nuc Blue kdorfman Thu, 07/14/2022 - 18:02

Hoechst type DNA stain for live cells

Nuc Blue protocol from Thermo Fisher

  • Culture cells in an appropriate medium and vessel for fluorescence microscopy.
  • Add two drops (20 uL = 1 drop) of NucBlue Live ReadyProbes Reagent per milliliter of medium.1
  • Incubate for 20 minutes, protected from light.
  • Image the cells.

  1. In some cases, more or fewer drops may be needed to achieve optimal staining intensity. Image quality may be improved by replacing the culture medium with Live Cell Imaging Solution (Cat. No. A14291DJ). [Kate says start with less, and work up. Rinse cells after incubation.] ↩︎

PS-Speck

PS-Speck kdorfman Thu, 02/01/2018 - 13:43

Fluorescent Beads

(Should also try MultiSpeck beads M 7901)

Protocol:

  • use polylysine slides so beads stick
  • 5 uL beads total (can mix colors on one slide)
  • 12 uL water; mix well
  • dry on a slide warmer (or hot plate at about 50C)
  • draw a circle on the bottom of the slide around the dried material
  • put 1 drop mounting medium over dried spot (gloppy! can't pipet!)
  • coverslip
  • nail polish

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

Phalloidin

Phalloidin kdorfman Thu, 08/13/2015 - 21:03

General protocol:

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

FITC phalloidin

FITC phalloidin kdorfman Fri, 06/01/2018 - 18:40

Thermo Fisher F432

From the manufacturer:

Fluorescein phalloidin is a high-affinity F-actin probe conjugated to the green fluorescent dye, fluorescein (FITC).

  • Selectively stains F-actin
  • Excitation/Emission: 496/516 nm
  • Superior to antibody staining
  • Optimal for fixed and permeabilized samples

10 and 20 uL aliquots

In histology freezer

Red phalloidins

Red phalloidins kdorfman Fri, 06/01/2018 - 18:42

Alexa Fluor 568 Phalloidin

Alexa Fluor 568 Phalloidin kdorfman Fri, 06/01/2018 - 18:41

Alexa Fluor 568 Phalloidin

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

In histology and bioimaging freezers

Rhodamine Phalloidin

Rhodamine Phalloidin kdorfman Fri, 06/01/2018 - 18:35

Thermo Fisher R415

From the manufacturer:

Rhodamine phalloidin is a high-affinity F-actin probe conjugated to the red-orange fluorescent dye, tetramethylrhodamine (TRITC).

  • Selectively stains F-actin
  • Excitiation/Emission: 540/565 nm
  • Superior to antibody staining
  • Optimal for fixed and permeabilized samples
  • Very widely cited fluorescent phalloidin conjugate

In 262A freezer

Texas Red-X Phalloidin

Texas Red-X Phalloidin kdorfman Fri, 06/01/2018 - 18:30

Thermo Fisher T7471

From the manufacturer:

Texas Red®-X phalloidin is a high-affinity F-actin probe conjugated to our bright, photostable, red fluorescent Texas Red®-X dye.

  • Selectively stains F-actin
  • Outstanding fluorescence performance
  • Excitation/Emission: 591/608 nm
  • Superior to antibody staining
  • Optimal for fixed and permeabilized samples

In histology freezer

Phloroglucinol

Phloroglucinol kdorfman Wed, 03/17/2021 - 16:10

Stains lignin

Phloroglucinol-HCl (Wiesner) Staining

Dissolve 0.3 g of phloroglucinol in 10 mL absolute ethanol to prepare a 3% phloroglucinol solution.

Mix one volume of concentrated HCl (37 N) to two volumes of 3% phloroglucinol in ethanol; this solution is phloroglucinol-HCl (Ph-HCl) or Wiesner stain. May 13, 2014

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4186213/

3% Phloroglucinol solution

to make: mL
at: %
mix: g phloroglucinol in 100% ethanol

Ph-HCL stain

to make: mL phloroglucinol stain
mix: mL 3% Phloroglucinol solution
with: mL HCl

Syto RNASelect

Syto RNASelect kdorfman Wed, 04/25/2018 - 19:47

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

TMRE

TMRE kdorfman Wed, 04/25/2018 - 21:17

Tetramethrylrhodamine ethyl ester perchlorate

mitochondria-specific Red fluophore

Biochemika (Sigma) 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

In freezers in 266A and 362A

Tetramethylrhodamine α-Bungarotoxin

Tetramethylrhodamine α-Bungarotoxin kdorfman Fri, 06/01/2018 - 19:06

Thermo Fisher T1175

Binds to acetylcholine receptor at neuromuscular junction

From the manufacturer:

Tetramethylrhodamine a-bungarotoxin can be used to visualize this receptor. Labeled a -bungarotoxin conjugates can be used to facilitate identification of nicotinic AChRs and to localize neuromuscular junctions.

Excitation⁄Emission (nm): 554⁄577

In freezer 266A

Toluidine blue

Toluidine blue kdorfman Wed, 03/17/2021 - 16:12

Toluidine blue is a basic thiazine metachromatic dye with high affinity for acidic tissue components.

It stains nucleic acids blue and polysaccharides purple and also increases the sharpness of histology slide images. It is especially useful today for staining chromosomes in plant or animal tissues

Toluidine Blue Stock Solution:

1g in 100 mL 70% ethanol

NaCl acid solution

1% NaCl aqueous, pH between 2 - 2.5 (w/ glacial acetic acid)

Working toluidine solution

1 part toluidine stock solution : 9 parts NaCl-Acid

Make this solution fresh and discard after use. pH higher than 2.5 will make staining less contrast.

To make: mL working toluidine blue solution,
mix: mL 1% NaCl + glacial acetic acid, pH ~2.25
with: mL 1% toluidine blue in 70% ethanol

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=&ved=2ahU…

Transferrin

Transferrin kdorfman Thu, 05/10/2018 - 14:49

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.