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Tissue clearing methods – Articles summary

tissue clearing methods for plants and animals

Tissue clearing methods – Articles summary

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tissue clearing methods for plants and animals

     When you want to image thick tissue samples with fluorescent proteins, the depth of imaging is limited by tissue transparency. To simplify, the level of transparency is the determining factor of how deep you can image. The more transparent the sample, the deeper you can image.

     The older methods used for rendering tissue transparent are mostly based dehydrating sample in a step by step manner and mounting them in high refractive index organic solvents. Other methods use chloral hydrate and Gum Arabica. Although these methods render tissue transparent, these are not compatible with fluorescent proteins.

     The tissue clearing methods that are listed below are compatible with fluorescent proteins. With these methods, you can image fluorescent samples 100s of microns deep that are otherwise difficult to image even 20-50 microns deep.

Plant tissue clearing methods

Picture1

An Optical Clearing Technique for Plant Tissues Allowing Deep Imaging and Compatible with Fluorescence Microscopy

Material used:

Arabidopsis thaliana: Leaf

Medicago truncatula: Leaf

Pisum sativum: Leaf and Root nodules

Nicotiana benthamiana: Leaf 

Zea mays: Leaf

Chemicals used:

6M Urea; Other reagents in the clearing solution are different for each tissue type. 

Time required for clearing:

48 hours to 3 weeks.

Dyes and/or Fluorescent proteins used

Citrine (yellow range)

Compatible with immunolabelling: Alexa 568

Calcofluor white (cell wall)

Refractive index of mounting medium:

NA

How deep tissue imaged:

Fresh samples 100 µm; Cleared tissue up to 350 µm.

Microscope used: 

Zeiss LSM 510 NLO multiphoton confocal microscope.  Zeiss LSM 710 confocal microscope.40X LD water immersion and 25X LD multi immersion (30% glycerol)

Conclusion:

I guess first of its kind in the plant clearing method. Now there are other methods that are faster in clearing (hours to days).

Take me to article

A Versatile Optical Clearing Protocol for Deep Tissue Imaging of Fluorescent Proteins in Arabidopsis thaliana

Material used:

Arabidopsis thaliana: Leaf, Root, Cotyledons, Isolated ovules, Siliques, Anthers and Shoot.

Chemicals used:

20-95% Thiodiethanol, Glycerol for mounting, 4%PFA for fixation. 

Time required for clearing:

1 hr to O/N fixation time + 1 - 3 hrs incubation in TDE. 

Dyes and Fluorescent proteins used

EGFP

Citrine (yellow range)

YFP

tdTomato

mCherry

Refractive index of mounting medium:

TDE (mounting medium) refractive index is adjustable up to 1.515 depending on the dilution.

How deep tissue imaged:

>240 microns with clearing method.

Microscope used: 

AxioImager Z.1         - 20X, 0.8NA

Leica TCS SP8            - 40X, 1.1NA W Corr

Olympus FV1200       - 40X

Zeiss LSM 780 NLO (2 photon) - 25x/0,8 Imm Korr and 40x/1,1 W Korr.

Conclusion:

This method clears tissue quicker than the Urea based method. The authors also showed the higher photostability of tdTomato in TDE. Despite the advantages mentioned above, tissue shrinkage may be a drawback in certain situations.

Take me to article

ClearSee: a rapid optical clearing reagent for whole-plant fluorescence imaging

Publication details:

Kurihara D et al. Development 2015

Material used:

Physcomitrella patens (Moss)

Arabidopsis thaliana: Leaf, Root, seedlings, pollinated  Pistil.

Chemicals used:

4%PFA for fixation; 

ClearSee:

Xylitol powder 10% (W/V),  sodium deoxycholate 15% (W/V), urea 25%(W/V) in water.

Time required for clearing:

Fixation time + 4 days to 4 weeks or more. 

Dyes and Fluorescent proteins used

mTFP1

sGFP

mGFP5

Venus 

mCitrine

YFP

mClover

tdTomato

mApple

mRFP1

Calcofluor White (cell wall stain)

Hoechst 33342

Refractive index of ClearSee:

1.41; For ClearSee V2 - 1.395.

How deep tissue imaged:

100-150 microns with clearing method + confocal laser scanning microscope.

400-670 microns with 2 photon microscope.

Microscope used: 

Olympus bx53: 10X

Nikon A1R MP (multiphoton): 25× WI, NA = 1.10, WD = 2.00 mm

Zeiss LSM780-DUO-NLO:  20× NA = 0.8

Storage of the cleared samples:

Cleared samples can be stored in the ClearSee reagent  5 (tested up to) months at room temperature.

Conclusion:

The most significant advantage of this method is the compatibility with fluorescent proteins (shown with ten different FPs) and also with cell wall staining (Calcofluor White) and nuclear stains(Hoechst 33342). The only drawback I can think of is the duration of tissue clearing (4 weeks in some cases). However, due to the advantages mentioned above waiting for 4 weeks may not be such a disadvantage.

Take me to article

TAE buffer (Tris-acetate-EDTA)

Tris-acetate-EDTA (TAE) is one of the most commonly used buffers for DNA and RNA Agarose electrophoresis. It has a lower buffering capacity compared to TBE (Tris-borate-EDTA) but runs nucleic acids faster, hence became the first choice. The composition that is currently being used is developed by the contribution of different research groups in the early 1970s. TAE is commonly prepared as a 50X solution with pH 8.5

Applications:

Commonly used buffer for DNA and RNA based methods using Agarose electrophoresis such as.

  • Restriction enzyme mapping.
  • Plasmid map confirmation.
  • To check the PCR (polymerase chain reaction) product and separation of correct size amplicons.
  • Quantification of DNA mixture (electrophoresis resolves DNA mixture and intensity of bands can be used of quantification).

pH:

No need to adjust the pH of the buffer. Adding the components in the specified quantities brings pH close to 8.5.

Composition

TAE buffer (Tris Acetate EDTA) composition
TAE buffer is used for DNA and RNA agarose electrophoresis. This table contains information for the preparation of 50X TAE ph8.5. There is no requirement for adjusting pH.
TAE buffer (Tris Acetate EDTA) composition
TAE buffer is used for DNA and RNA agarose electrophoresis. This table contains information for the preparation of 50X TAE ph8.5. There is no requirement for adjusting pH.
Reagent Molecular weight 1X TAE molarity 50X TAE molarity Add for 500ml of 50X TAE Add for 1L of 50X TAE
Tris Base 121.14 g/mol 40 mM 2 M 121.1 g 242.2 g
Acetic Acid (glacial) 60.5 g/mol 20 mM 1 M 30.25 ml 60.5 ml
EDTA Sodium salt dihydrate 372.24 g/mol 1 mM 50 mM 9.3 g 18.6 g

Glacial acetic acid: Glacial is a fancy term used to mention acetic acid which has very less amount of water in it (consider less than 1% for the sake of discussion). Vinegar is also acetic acid but not glacial since it contains water ranging from 80-95%.

Reagents:

  • Tris Base
  • Acetic acid (glacial)
  • Disodium EDTA
  • Distilled water

Materials and instruments:

  • Glass Beaker
  • Weighing balance
  • Magnetic stirrer and pellet
  • pH meter
  • Measuring cylinders

Procedure:

  • Add the components in half the volume of ddH2O intended for the volume.
  • Put the beaker in magnetic stirrer with a magnetic pellet.
  • EDTA dissolves very slowly, so have a cup of coffee and relax.
  • Once all components dissolved, make up the volume with ddH2O intended for.
  • Now you have 50X TAE.
  • For making 1X TAE from 50X stock, add one part 50X TAE to 49 parts of ddH2O.

Caution:

Storage:

Store the TAE buffer at room temperature.

References:

wikipedia

History and principles of conductive media for standard DNA electrophoresis.

LB broth / LB medium

Giuseppe Bertani formulated LB broth in 1951 in an attempt to optimise Shigella growth and plaque formation. Although LB media is known as “Luria Broth”, “Luria-Bertani” medium, or “Lennox Broth”, it was initially named as “Lysogeny Broth”. LB broth’s agar form should be designated LA but now referring as LBA/LB. There are many formulations available for LB medium, in this article, we are providing most used formula (based on CSH protocols).

Applications

  • LB is a rich medium, used as general purpose bacterial culture medium especially Enterobacteriaceae members (E.coli is one among them).
  • Lysogeny broth is also used for coliphage plaque assays.

pH

Cold Spring Harbor Protocol recommends pH to be 7.0. However, depending on the application pH can be 7.0 – 8.0. Adjust the pH with 1N NaOH and 1N HCl. Although the protein/peptide component of the medium shows some degree of buffering capacity, which is not sufficient for rapidly growing bacterial population. For general purposes, this is not an issue. However, some labs prefer to make LB in 5-10mM TRIS buffer of the desired pH.

Composition

LB broth / LB medium
Adjust the pH to 7.0 with 1 N NaOH and 1N HCl.
LB broth / LB medium
Adjust the pH to 7.0 with 1 N NaOH and 1N HCl.
Reagent For 100 mL For 500 mL For 1000 mL
Tryptone 1 g 5 g 10 g
NaCl 1 g 5 g 10 g
Yeast extract 0.5 g 2.5 g 5 g
Agar 1.5 g 7.5 g 15 g

Reagents

Along with the reagents mentioned in the table 1N NaOH.
1N HCl.

  • 1N NaOH.
  • 1N HCl.

Materials and instruments

  • Glass Beaker.
  • Conical flasks.
  • Cotton plugs.
  • pH Meter.
  • Pipettes and tips.
  • Petri dish.
  • Weighing balance and paper boats.
  • Magnetic stirrer and pellet.

Procedure

  1. Determine the volume to be made and weigh the reagents accordingly (The precalculated values in the table for specific volume might be helpful).
  2. Add the reagents to a glass beaker and make up the volume to 90% of planned with ddH2O (e.g., 900 ml if you have planned for 1L broth). DO NOT add agar (if preparing LB medium with agar) at this point.
  3. Dissolve the components in the beaker using magnetic stirrer.
  4. Adjust the pH to the desired value. (see the pH section above for details).
  5. Adjust the broth to final volume using ddH2O.
  6. Transfer the broth to conical flask or aliquot into smaller volumes.
  7. Add agar if planned to make LB medium with agar. Adding agar after aliquoting will ensure equal distribution.
  8. Close the mouth of the flask with a cotton plug. Seal it further with paper and rubber band.
  9. Autoclave for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle.

Storage

Store broth at room temperature.

Caution

  • Opening and closing of media should be done in aseptic conditions only. Opening in the open air might lead to contamination.
  • Adding agar before adjusting pH might result in mild hydrolysis.

References

PBST Buffer (PBS with Tween 20)

PBST or PBS-T (phosphate buffered saline with Tween 20) is PBS buffer with detergent such as Tween 20 or Triton X-100 (CSH Protocols recommends Tween 20). Detergent concentration can vary depends on requirement but generally vary from 0.05 – 2% (CSHP recommends 0.05). We are giving composition based on CSHL protocols in the tabular form.

Applications

Used as washing solution for:

  1. Western blot membranes.
  2. PFA (Paraformaldehyde) fixed samples.
  3. Microtiter plate wells in ELISA assays.
  4.  Immunohistology.

pH

Keep pH at 7.4 with HCl and NaOH.

Composition

PBS-T composition
0.05% (V/V) Tween 20. pH 7.4.
PBS-T composition
0.05% (V/V) Tween 20. pH 7.4.
1X PBS 500 mL 1X PBS 1L 10X PBS 500 mL 10X PBS 1L
Tween 20 to be added 25 µl 50 µl 250 µl 500 µl

Reagents required

  1. 1X PBS (Click here for composition).
  2. Tween 20.

Material and instruments required

  1. Pipettes and tips.
  2. Glassware for mixing
  3. Measuring cylinder

Procedure

  1. Make 1X PBS buffer
  2. Add Tween-20 as desired concentration
  3. Swirl slowly until dissolved

Storage

Store at room temperature. Keeping in lower temperature might lead to crystallization.

Caution

Tween 20 and Triton X-100 are viscous liquids. You will have to cut the narrow end of the pipette tip for easy aspiration.

If you find pipetting still difficult, weigh the detergent (consider density for determining volume) and add PBS to it.

Detergents make froth upon agitation. Swirl gently to avoid froth.

References

  1. Cold Spring Harbor Protocols
  2. Openwetware

4% Paraformaldehyde (PFA) solution preparation

Paraformaldehyde (PFA) in PBS is one of the widely used fixatives for Immuno-histochemistry (IHC) and fluorescent protein labelled samples. Paraformaldehyde is a polymer of formaldehyde with a wide range of monomeric units typically 8-100. PFA does not have the capacity to fix samples, hence it must be depolymerised in the solution. Heating the PFA powder in the solution leads to its depolymerisation. Although 4% PFA is widely used, there are circumstances where it is used as low as 0.5% to as high as 16%. When dissolved, paraformaldehyde breaks into formaldehyde in solution. Formaldehyde fixes (halts) metabolism by cross-linking protein molecules especially with lysine. It is important to note, that formaldehyde-based fixation is too slow and may take from a few hours to days to fix samples. PFA is recommended to be made in 1X PBS buffer.

Below, we have provided a step by step process to prepare 4% PFA solution.

Applications

  • Fixation solution for Immuno-histochemistry and fluorescent protein labelled samples.

pH

Adjust pH 6.9 to 7.4 depending o application with 1N HCl and 1N NaOH.

Composition

4% Paraformaldehyde (PFA) fixative composition and preparation
Keep the pH between 7.2 to 7.4 using HCl and NaOH. 0.5% Triton X-100
4% Paraformaldehyde (PFA) fixative composition and preparation
Keep the pH between 7.2 to 7.4 using HCl and NaOH. 0.5% Triton X-100
Reagent Add for 100 ml Add for 500 ml Add for 1 L
Paraformaldehyde 4 g 20 g 40 g
1X PBS 80 ml + 20ml 400 ml + 100 ml 800 ml + 200 ml

Reagents required:

Material and instruments required:

  • Glass Beaker.
  • Filter unit.
  • Hotplate magnetic stirrer and a magnetic bead.
  • Ventilation hood.
  • Pipettes and Tips.
  • pH meter.
  • Thermometer.

Procedure:

Here we are describing steps for 4% PFA of the 1L solution. The same trend follows for other volumes as well.

  1. Take 800 mL of 1X PBS.
  2. Add 40 g of Paraformaldehyde powder to 1X PBS.
  3. Stir the mixture at 60˚C in ventilation hood (DO NOT Boil).
  4. PFA powder does not dissolve instantly, you need to raise the pH of the mixture by adding 1N NaOH drop by drop until a clear solution is formed.
  5. There may be small undissolved particles. Cool the solution to room temperature and filter to remove particles.
  6. Adjust the volume to 1L with 1X PBS.
  7. Add 0.5% Triton X-100
  8. Check the pH and adjust with HCl and NaOH if required.
  9. Aliquot into small volumes.

Storage

At room temperature, for 1-2 weeks. At 4oC for a few weeks. For long term storage (up to a year) at -20o C.

References

  1. Cold Spring Harbor Protocols.
  2. BOSTER.

Phosphate Buffered Saline (PBS)

Phosphate buffered saline (a.k.a PBS or PBS buffer) is one of the most used buffers since it is isotonic to most of the cells. We have placed reagents required for preparation in a table. This table also contains information on molarity of each reagent and amount of salt (s) to be added for making 500mL and 1L of 1X and 10X. For your convenience, we have given the molecular weights of each reagent and for it’s most used hydrated forms too.  It is up to the user to determine whether hydrated forms make any difference for the experiments or not.  

There are many different formulations for phosphate buffered saline. Some of them contain calcium and magnesium salts. In this page, we are restricting ourselves to provide the most used PBS composition based on (CSHL protocols and Molecular cloning by Sambrook).

Applications

  • Washing buffer for cells/tissue.
  • Diluent.

pH

Adjust pH to 7.2 or 7.4 (depending on requirement) using 1N HCl and 1N NaOH

Composition

Phosphate buffered saline (PBS) composition and preparation
pH of the buffer to be adjusted to 7.4 with HCl and NaOH.
Phosphate buffered saline (PBS) composition and preparation
pH of the buffer to be adjusted to 7.4 with HCl and NaOH.
Reagent M.W Molarity of 1X Add for 500 ml 10X Add for 1 L of 10X Add for 500 ml 1X Add for 1 L of 1X
NaCl 58.44 137 mM 40 g 80 g 4 g 8 g
KCl ‎74.54 2.7 mM 1 g 2 g 0.1 g 0.2 g
Na2HPO4 141.96 10 mM 7.2 g 14.4 g 0.72 g 1.44 g
Na2HPO4 2H2O 177.99 10 mM 8.9 g 17.8 g 0.89 g 1.78 g
Na2HPO4 7H2O 268.07 10 mM 13.4 g 26.8 g 1.34 g 2.68 g
Na2HPO4 12H2O 358.14 10 mM 17.9 g 35.8 g 1.79 g 3.58 g
KH2PO4 136.08 1.8 mM 1.2 g 2.4 g 0.12 g 0.24 g

Instruments and other requirements

  • Weighing balance.
  • Graduated cylinder and a glass beaker.
  • Magnetic stirrer and magnetic bead/pellet.
  • pH meter.
  • 1N HCl and 1N NaOH for pH adjustment.

Procedure

Use ddH2O only for making PBS.

  1. Determine how much volume (500mL or 1L or any other) and concentration (1X or 10X) you want to make.
  2. Weigh the reagents accordingly (Refer to the table above).
  3. Add salts one after the other to the ddH2O (take 400ml or 800ml of initial volume if you want to make 500mL or 1L respectively).
  4. Dissolve salts by using magnetic stirrer.
  5. Adjust pH to 7.4 using HCl and NaOH.
  6. Bring the volume to 500mL or 1L as planned.
  7. Aliquot to smaller volumes if needed (frequent opening and closing in open air might result in microbial growth).
  8. Autoclave for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle or Filter sterilize.

Storage

       You can store PBS (Phosphate buffered saline) at room temperature. Storage of 10X at 4˚C might lead to the formation of crystals.

Reference

Cold Spring Harbor Protocols.

Arabidopsis Pollen Germination Medium (Leonor 2007)

There are a handful of Pollen Germination Media (PGM) formulated for one of the widely used model plant, Arabidopsis. In this article, we have provided composition of one of the robust PGM. This PGM composition is adopted from a publication by Leonor 2007 (see the reference at the end of the article). If you are a beginner, I highly recommend you to read the full article since it shows the effect of temperature, light, pH and other factors on pollen germination and pollen tube growth. We have placed reagents required for preparation in a tabular form. This also contains information on Molarity of each reagent and their quantities to be added for making 10 mL of stock solution. We have also provided the information about different dilutions that can be made from the stock.

For your convenience, we have given the molecular weights of each reagent and for its most used hydrated forms too.  It is up to the user to determine whether hydrated forms make any difference for the experiments or not.  

pH

The pH of the medium should be adjusted to 7.5 using dilute HCl and KOH.

Composition

pH of the medium is one of the critical factor that determines germination efficiency. For this medium, pH should be 7.5. Achieve this pH using HCl and KOH.
pH of the medium is one of the critical factor that determines germination efficiency. For this medium, pH should be 7.5. Achieve this pH using HCl and KOH.
Reagent M.W Working concentration Stock solution Add for 10 ml stock Add from stock for 1X of 50ml
Sucrose 342.3 10% - - 5 g
H2BO3 61.8 0.01% 1% 0.1 g 0.5 ml
CaCl2(anhydrous) 110.98 5mM 1M 1.1 g 250 µl
CaCl2 .2H2O 147.01 5mM 1M 1.4 g 250 µl
KCl 74.5 5mM 1M 0.745 g 250 µl
MgSO4 120.36 1mM 1M 1.2 g 50 µl
MgSO4 .7H2O 246.47 1mM 1M 2.465 g 50 µl
Noble Agar - 1.50% - - 0.75 g

Reagents

  • Sucrose
  • Boric acid
  • Calcium chloride
  • Potassium chloride
  • Magnesium sulphate
  • Noble agar
  • Distilled water
  • HCl
  • KOH

Instruments and other requirements

  • Weighing balance. paper boats.
  • Graduated cylinder and a glass beaker.
  • pH meter. Magnetic stirrer and magnetic bead/pellet.
  • pipette.
  • Tissue paper.

Procedure

Use ddH2O only for making PGM

You can choose to make 1X (working concentration) or make stocks and use them to make 1X whenever you want. Making stock solutions eliminate the possibility of errors that may happen while measuring very low quantities of reagents. Making working concentration from stocks also helps you to add some other reagents without further diluting working concentration.  

  1. Determine whether you want to start with stock solutions or directly making working concentration.
  2. Weigh the reagents accordingly (Refer to the table above).
  3. Dissolve salts by using magnetic stirrer or vortex mixer.
  4. Adjust pH to 7.5 using HCl and KOH (pH is one of the critical factors)
  5. Make the volume as planned.
  6. Sterilization of media is not required for immediate use. However, doing filter sterilization might increase its shelf life by preventing microbial growth.
  7. Aliquot to smaller volumes if needed (frequent opening and closing in open air might result in microbial growth).
  8. Noble agar is preferable over agar as solidifying agent.

Storage

Store PGM (Pollen Germination Medium) stock solutions at 4˚C for 1-2 weeks.

Reference

Temperature as a determinant factor for increased and reproducible in vitro pollen germination in Arabidopsis thaliana.