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.

Materials required but not in kit:

• PBS

• 3.7% formaldehyde in PBS

• 0.5 % Triton-X in PBS

• 3% BSA in PBSTX

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)

DAPI Fluoromount Fisher OB010020

Store at room temp, in the dark

Aliquot 1 mL into dark tube.

Keep in "mounting" drawer in 360.

Make droppers by melting the end of a glass Pasteur pipet to make a ball.

1 mg/mL DAPI stock solution from Rolf

1 uL per 10 mL 25% glycerol

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 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.

• remove growth medium

• rinse with HBSS

• 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 with warm HBSS

• put warm non CO2 medium in dish

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 (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.

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 HBSS (Hank's)(0.34 mg/mL BSA) to 10 µL in tube

Makes enough for 8 groups.

Aliquot ~120 µL per group, enough to cover the small circular coverslip in the viewing dish.

To use:

• Rinse with HBSS (see notes)
• Treat with ceramide solution
• incubate in cold 30 min
• rinse 3x in cold HBSS
• replace HBSS with non-CO2 medium, incubate ~20 minutes

NOTES:

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

• If following with ER tracker, rinse with HBSS, then replace with warm non-CO2 medium

• If following with Mitotracker, rinse with PBS, then replace with warm non-CO2 medium

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.

CellLight^TM Nucleus-RFP

Invitrogen (Thermo-Fisher) C10603

1 mL - received 10/26/23 - purchased for Bioimaging

Store in refrigerator

manual

Simplified protocol:

Starting concentration = 10⁸ particles per mL

Working concentration = 10 - 50 particles per cell

Use low-passage number cells;

estimate number of cells at time of treatment - should be no more than 70% confluent

(number of cells x ~30 particles/cell)/10⁸ particles/mL = mL CellLight to use

Mix thoroughly but gently with cell medium

Image cells after about 16 hours

To stain	cells
at	particles per cell (between 10 & 50)
add	µL Nucleus-RFP

From the mfgr:

CellLight Nucleus-RFP, BacMam 2.0, provides an easy way to label nuclei with red fluorescent protein (RFP) in live cells. Simply add the reagent to your cells, incubate overnight, and the cells are ready to image in the morning.

This ready-to-use construct is transfected into cells using BacMam 2.0 technology, where it expresses RFP fused to the SV40 nuclear localization sequence. You can observe nucleus-RFP behavior in live cells without the cellular toxicity associated with intercalators and label with multiple tracking or tracing dyes to image dynamic cellular processes.

Cells expressing CellLight constructs can also be fixed with formaldehyde for multiplexed imaging using immunocytochemical techniques.

CellLight Technology is:

• Fast and convenient: simply add CellLight reagent to your cells, incubate overnight, and image—or store frozen, assay-ready cells for later use
• Highly efficient: up to 90% transduction of a wide range of mammalian cell lines, including primary cells, stem cells, and neurons
• Flexible: co-transduce more than one BacMam reagent for multiplex experiments or co-localization studies; tightly control expression levels by simply varying the dose
• Less toxic: CellLight reagents are non-replicating in mammalian cells and are suitable for biosafety level (BSL) 1 handling

BacMam Technology

CellLight Nucleus-RFP, BacMam 2.0, is a fusion construct of SV40 nuclear localization sequence and TagRFP, providing accurate and specific targeting to cellular nucleus-RFP. This fusion construct is packaged in the insect virus baculovirus, which does not replicate in human cells and is designated as safe to use with biosafety level (BSL) 1 in most laboratories. BacMam technology ensures that most mammalian cell types are transduced/transfected with high efficiency and minimal toxicity. This transient transfection can be detected after overnight incubation for up to five days—enough time to carry out most dynamic cellular analyses. Like any transfection/transduction technique, the BacMam method does not transfect/transduce all of the cells with equal efficiency, making it poorly suited to cellular population studies or automated imaging/counting. CellLight reagents are ideal for experiments where cellular or subcellular co-locatization is required, or for cellular function studies that need special resolution.

Visualize staining your cell without wasting your reagents, antibodies, or time with our new Stain-iT Cell Staining Simulator.

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

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

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 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

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

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 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 for Madelaine's plant anatomy class

• 1 % Toluidine blue
• 0.5% borax

---

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…

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.

http://fscimage.fishersci.com/msds/89336.htm