Digital Assignment

NEUROBIOLOGY AND
COGNITIVE SCIENCE
APOORVA MITTAL 16BBT0006
TOPIC:
CELLULAR STAINING
Haematoxylin and Eosin Staining
This 5 µm paraffin section of rat intestine was stained with hematoxylin (blue) and eosin Y (red).
Technique
These are two stains used in the examination of thin slices of biological tissue. Contrast is
created by the stains where Haematoxylin turns the nuclei blue while eosin turns the cytoplasm
as well as other parts pink or red.
Preparation
Haematoxylin
1. Measure 10 grams of haematoxylin crystals 500 ml of water (70- 80 degrees centigrade) and
mix to dilute completely
2. In a separate flask, measure 20 grams of alum and mix with 500 ml of hot tap water (70- 80
degrees)
3. Mix the two mixtures together (1 and 2)
4. Add 1 gram of crystal.
Whereas alum is the mordant, thymol prevents fungal growth.
5. The mixture is then kept in a translucent flask away from direct sunlight for one week. This
is covered with a paper towel that allows for air circulation (early maturation). The solution is
then put in to a dark flask and topped tightly after one week, and stored in a dark place for 3
weeks. (Late maturation)
Eosin (1000ml)
Measure 10 grams of eosin crystals and add to mix in 1000ml of hot tap water (70- 80 degrees).
This should be mixed to dilute and stored in a dark flask. This can be used directly.
Hematoxylin (hematin) binding to DNA in the cell nucleus.
Procedure
1. A rehydrated section is stained in a solution of haematoxylin for 20 to 40 minutes
2. The section is then washed in tap water for about 3 minutes until it turns blue,
3. The section is the differentiated in 70percent ethanol that contains 1 percent of HCL for
about 5 seconds to remove excess dye and allow the nuclear to emerge,
This is then washed in tap water,
5. Stain with eosin for 10 minutes,
6. Then wash for about 1 to 5 minutes in tap water,
7. Dehydration, clear and mount on a rack.
Acid and Basic Fuchsin Stain

Technique
Acid fuchsin is a magenta red acid dye that is largely used for plasma staining whereas
basic fuchsin is a magenta basic dye largely used to stain the nucleus.
The technique is also referred to as acid fast staining. The acid fast bacteria have a waxy
substance (mycolic acid) on their cell wall that makes them impermeable to staining
procedures.
The term acid fast is used since they resist decolourization with acid alcohol. Carbol fuchsin,
the primary stain contains phenol, which helps solubilize the cell wall whereas heat is used to
increase the penetration of the stain.
On using alcohol to decolorize, cells will be decolorized except for acid fast ones. Methylene
blue is used as the counterstain to counterstain any cell that was decolorized. At the end of the
procedure, acid fast cells remain red/pink while non-acid fast cells retain a blue color.
Preparation
Preparation of carbol fuchsin by mixing two solutions:

Solution 1- 0.2 grams of basic fuchsin and 10 ml of 95 percent ethanol,
Solution 2- 5 grams phenol and 90 ml of distilled water,
Procedure
1. Swab pulp larvae together so as to allow the pulp to spread over the slide and allow to dry
2. Heat fix by flaming over a burner several times,
3. Flood using 0.2 percent of carbol fuchsin for about 30 seconds,
4. Wash off the stain and then air dry/ gently blot dry before examination.
DAPI - 4′,6-diamidino-2-phenylindole
The blue-fluorescent DAPI nucleic acid stain preferentially stains dsDNA; it appears to
associate with AT clusters in the minor groove. Binding of DAPI to dsDNA produces a ~20-
fold fluorescence enhancement, apparently due to the displacement of water molecules from
both DAPI and the minor groove. DAPI also binds RNA, however in a different binding
mode—one thought to involve AU-selective intercalation. The DAPI/RNA complex exhibits a
longer-wavelength fluorescence emission maximum than the DAPI/dsDNA complex (~500 nm
versus ~460 nm) and a quantum yield that is only about 20% as high.
Drosophila melanogaster embryo staining.
Preparing the DAPI stock solution
To make a 5 mg/mL DAPI stock solution (14.3 mM for the dihydrochloride or 10.9 mM for
the dilactate), dissolve the contents of one vial (10 mg) in 2 mL of deionized water (dH 2O) or
dimethylformamide (DMF). The less water-soluble DAPI dihydrochloride may take some time
to completely dissolve in water and sonication may be necessary.
Adherent cells for fluorescence microscopy
Sample preparation
Use the fixation protocol appropriate for your sample. DAPI staining is normally performed
after all other staining. Note that fixation and permeabilization of the sample are not necessary
for counterstaining with DAPI.
Counterstaining protocol
1. Equilibrate the sample briefly with phosphate-buffered saline (PBS).

2. Dilute the DAPI stock solution to 300 nM in PBS. Add approximately 300 µL of this dilute
DAPI staining solution to the coverslip preparation, making certain that the cells are
completely covered.
3. Incubate for 1–5 minutes.
4. Rinse the sample several times in PBS. Drain excess buffer from the coverslip and
mount. We recommend using a mounting medium with an antifade reagent such as
our SlowFade Gold antifade reagent or ProLong Gold antifade reagent.
5. View the sample using a fluorescence microscope with appropriate filters.
Hoechst Staining
Protocol
Preparing Hoechst dye stock solution
1. Prepare the Hoechst dye stock solution by dissolving the contents of one vial (100 mg)
in 10 mL of deionized water (diH2O) to create a 10 mg/mL (16.23 mM) solution. Note:
Hoechst dye has poor solubility in water, so sonicate as necessary to dissolve. The 10
mg/mL Hoechst stock solution may be stored at 2–6°C for up to 6 months or at ≤–20°C
for longer periods.
Labelling cells
1. Culture cells in an appropriate medium and vessel for

fluorescence microscopy.
2. Prepare the Hoechst staining solution by diluting the
Hoechst® stock solution 1:2,000 in PBS.
3. Remove the medium.
4. Add sufficient staining solution to cover the cells.
5. Incubate for 5–10 minutes, protected from light.
6. Optional: You may image directly in the staining

solution, if you wish.
7. Remove the staining solution.
8. Wash the cells 3 times in PBS.
9. Image the cells.

Neutral Red Staining
Neutral Red is a weak cationic azine dye that is used extensively as a nuclear stain in a variety
of biological stain applications. This Neutral Red Staining Solution is can also be used as a
counterstain in combination with other dyes. The Staining Solution is suitable for tissue or cells
fixed, not for living cells. Neutral Red is a pH indicator as well, changing color from red to
yellow over the pH range 6.8~8.0.
Procedure
1. Sample preparation:
a) For cells in culture: Fix cells with 4% paraformaldehyde for 10~20 minutes, and
rinse twice with distilled water for 2~5 minutes.
b) For frozen sections: Rinse the sections with distilled water for 2~5 minutes.
c) For paraffin sections: Dewax and rehydrate tissue section according to standard
protocols. Rinse with distilled water for 2 minutes.
2. Neutral Red staining: Add Neutral Red Staining Solution (Component A) and incubate
for 2~10 minutes at room temperature. (Note: Each cell line should be evaluated on an
individual basis to determine the optimal time to stain depending upon fixation and cell
line.)
3. Wash twice with distilled water, 2 minutes each. Optional: after wash with distilled
water, sample can be dehydrated in alcohol and cleared in xylene and mounted
according to standard protocol.
4. Samples can be observed under microscopy.

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NEUROBIOLOGY AND

APOORVA MITTAL 16BBT0006

3. Mix the two mixtures together (1 and 2)

4. Add 1 gram of crystal.

Whereas alum is the mordant, thymol prevents fungal growth.

Hematoxylin (hematin) binding to DNA in the cell nucleus.

1. A rehydrated section is stained in a solution of haematoxylin for 20 to 40 minutes

This is then washed in tap water,

5. Stain with eosin for 10 minutes,

6. Then wash for about 1 to 5 minutes in tap water,

7. Dehydration, clear and mount on a rack.

Acid and Basic Fuchsin Stain

Preparation of carbol fuchsin by mixing two solutions:

Solution 2- 5 grams phenol and 90 ml of distilled water,

2. Heat fix by flaming over a burner several times,

3. Flood using 0.2 percent of carbol fuchsin for about 30 seconds,

Drosophila melanogaster embryo staining.

Preparing the DAPI stock solution

Adherent cells for fluorescence microscopy

1. Equilibrate the sample briefly with phosphate-buffered saline (PBS).

Preparing Hoechst dye stock solution

1. Culture cells in an appropriate medium and vessel for

3. Remove the medium.

4. Add sufficient staining solution to cover the cells.

5. Incubate for 5–10 minutes, protected from light.

6. Optional: You may image directly in the staining

7. Remove the staining solution.

8. Wash the cells 3 times in PBS.

9. Image the cells.

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