EXERCISE 4C: FLOATING OUT,
STAINING, MOUNTING AND
LABELLING OF STAINED
SECTIONS
ROUTINE TISSUE PROCESSING
Exercise 4a
FLOATING
Floating out of tissue ribbons begin once
you already have your serial sections.
Sections are removed from the
microtome and the ribbons are placed in
the tissue floatation bath.
FLOATING
1. Temperature of the floatation bath
 maintained at 45-50°C
 approx. 6-10°C lower than the
melting point of paraffin wax
 Normally, the paraffin wax with a
melting point of 56°C is used for
routine work.
FLOATING
Make sure that the water is free of any
debris that can interfere with the
procedure.
Some floatation baths are equipped with
LED lights to allow easy visualization of
such occurrences
Gliding an absorbent cloth over the water
surface is enough to rid certain particles.
FLOATING
2. Float ribbon on the floatation
bath
One end of the ribbon is lowered first.
‣ The remainder of the ribbon is lowered
gradually with a slight pull.
‣ If done so, there are fewer tendencies
for air bubbles to form under the
section
FLOATING
 To remove wrinkles, gently tease
with a camel’s hair brush or
forceps.
Thirty seconds should be long enough
for a ribbon to flatten.
‣ Using curved forceps on either
side of the wrinkle, gently pull the
wrinkle apart.
‣ It may be necessary to cut another
ribbon
 To remove bubbles, place a pair of
fine-eyed forceps beneath the
ribbon on the floatation bath.
 For circular structures like an eye,
place the section on the slide that
has been pre-flooded with 50%
alcohol. The slide is gently
immersed in the floatation bath
and the section of the eye will float
on the surface
FLOATING
3. Separate the sections
 Using a pair of curved forceps,
separate the sections from each
other by applying gentle pressure
at the junction of the section.
 Pick up sections using the
microscope slide to separate
them.
 In case of serial sections, do not
separate more than one section
(or block of sections) at a time.
 Place sections with the smooth
(shiny) side down.
FLOATING
4. Mount on glass slides
Using a clean, dust-free slide, insert the
sections into the floatation bath
perpendicularly
 Place the sections at the middle of
the slide to allow free margins for
 subsequent cover-slipping and
labeling
To do the following, dip the entire slide
halfway through.
The section will adhere to the area of the
slide where the surface of the water is.
The latter portion of the section will
attach further down the slide.
FLOATING
 Label with pencil the frosted end
of the slide.
 The accession number is taken
from the cassette (block).
FLOATING
 Sections on slides should be
drained before the final drying on
the hot oven. This can be done by
placing the slide perpendicularly
on paper towel to absorb the
water.
 After draining, the slides can be
placed in a slide rack
FLOATING
5. Place the slide rack in the drying oven
to dry the slides and melt the paraffin
The small amount of water under the
section will allow further flattening when
heat is applied to dry the section.
 The temperature should be the
melting point of the paraffin.
FLOATING
Other methods of drying include:
- leaving the slides in a 37°C
incubator overnight (for delicate
specimens), or
- by drying on a stretching table at
45° to 55°C for 30 to 45 minutes
FLOATING
6. Cool the slides to room temperature
before proceeding to staining.
*ADHESIVES
Mayer’s Egg Albumin is used as an
adhesive wiped onto the glass slide to
allow the tissue section to adhere it.
Mayer’s egg albumin is prepared by
mixing equal parts egg white, distilled
water and glycerin. A crystal of thymol
is added to prevent mold formation
STAINING
The end product of optimal tissue
processing and successful microtomy is a
thin and transparent tissue section set on
a slide. The only way to render the tissue
visible under the light microscope is to
provide color to the cell components
through stains.
Differential staining enables microscopic
evaluation of all areas of the tissue. For
clinical laboratories, such evaluation is
necessary for the anatomic pathologist to
render a histopathology diagnosis.
The most common “routine” staining
is the hematoxylin and eosin (H&E)
method.
STAINING
The H&E stain displays a wide range of
cellular features.
Hematoxylin has a deep blue-purple
color and stains nucleic acids by a
complex, incompletely understood
reaction.
Eosin is pink and stains proteins non-
specifically.
In a typical H&E stained section, nuclei
appear blue while the cytoplasm and
extracellular matrix show different
shades of pink.
STAINING
Nuclear chromatin activity influences the
varying depth of hematoxylin stain.
Nucleoli stain with eosin.
Ribosome-rich cytoplasm appears blue-
tinged. A prominent Golgi zone shows
perinuclear absence of staining.
STAINING
STEP 1: DEPARAFFINIZATION
Dewaxing or deparaffinization starts with
heating the slides in a 60°C oven for at
least 30 minutes to soften the wax.
Some tissues benefit from 40°C
overnight (i.e. bone and brain sections)
Sections inadequately dried will not
deparaffinize sufficiently with xylene and
will not pick up the stains consistently.
The end result will be poorly H&E stained
sections
STEP 1: DEPARAFFINIZATION
Deparaffinization:
The process in which paraffin is removed
from the sections.
Xylene is not miscible with water so
sections must be dried before being
placed in xylene.
Section drying and paraffin removal are
critical to successful H&E staining.
STEP 1: DEPARAFFINIZATION
The uneven staining in this section is a
direct result of not enough time in in
xylene to remove all of the paraffin from
the tissue. This artifact is also seen when
the support media for frozen sections has
not been adequately remove.
STEP 2: REHYDRATION
100% Reagent Alcohol / Absolute
Ethanol: Is the intermediary between
xylene and hydration.
Removal of the xylene with 100% alcohol
before hydration is critical to a successful
H&E stain.
STEP 2: REHYDRATION
95% Alcohol to tap water:
Hydration begins the gradual
introduction of water to the tissue
sections.
STEP 2: REHYDRATION
Tap water - Wash until oily appearance in
slides disappear
Tap water may be substituted with PBS
(phosphate buffer solution)
Washing ensures that all alcohol is
removed, and the tissue specimen is fully
rehydrated.
STEP 2: REHYDRATION
Slide above was not rehydrated properly
and contained trace amounts of alcohol
(or even xylene/wax).
This caused uneven staining
STEP 3: NUCLEAR STAIN
Hematoxylin is used as to stain nuclear
components. Many types exist depending
if the staining procedure involved is
progressive staining or regressive
staining.
Common regressive hematoxylin stains:
- Harris’
- Delafield’s
- Erlich’s
Common progressive hematoxylin stains:
- Mayer’s
- Gill’s I, II
STEP 4: DIFFERENTIATION
On most occasions when Harris
hematoxylin (or other regressive stains)
is employed, a differentiation
(destaining) step is required to remove
non-specific background staining and to
improve contrast. A weak acid alcohol is
used.
After this treatment, blueing and
thorough rinsing is again required.
Staining methods that include a
destaining or differentiation step are
referred to as “regressive” stains.
- 0.5% Acid alcohol is used as a
differentiating agent.
STEP 4: DIFFERENTIATION
Dip in tap water a few times (5 dips)
This is mainly done to remove excess
acid alcohol
STEP 4: DIFFERENTIATION
ABOVE: Muscle tissue, normal stained
Below: Muscle tissue, no differentiation
STEP 5: BLUING
After rinsing in tap water, the section is
“blued” by treatment with a weakly
alkaline solution.
Bluing reagents change the reddish –
purple hematoxylin to a blue or purple
blue color. It is a pH dependent reaction
and occurs in an alkaline solution.
- Lithium carbonate - 10–30
seconds or until dark blue color
develops. Do not agitate the slides
when in the blueing agent to
prevent section loss.
STEP 5: BLUING
Dip in running tap water (15 dips)
This is to remove excess bluing agent and
to stop to bluing process to prevent
overbluing.
The section can now be rinsed and
checked to see if the nuclei are properly
stained, showing adequate contrast and
to assess the level of background stain.
STEP 5: BLUING
a. In this section the epidermal
nuclei are poorly defined and are
pinkish in color. This section was
not properly “blued” in alkaline
water after hematoxylin staining
(skin, H&E).
b. This shows another section that
was properly “blued” after the
nuclear stain. Here the nuclei are
much better defined (skin, H&E).
STEP 6: COUNTERST AINING
Eosin in the H&E procedure is referred
to as a counterstain. It stains nearly
everything that hematoxylin will not
stain.
When applied correctly, eosin produces
three different hues which can be used to
differentiate various tissue elements; red
blood cells stain dark reddish orange,
collagen stains a lighter pastel pink, and
smooth muscle stains bright pink.
STEP 6: COUNTERSTAINING
Inefficient washing after “blueing”
(leaving residual alkali) causes eosin
staining to be weak and uneven.
STEP 6: COUNTERSTAINING
A section of lung stained H&E. The
eosin stain is uniformly very weak and
quite unacceptable. Note that the only
components stained with eosin are the
red blood cells.
STEP 7: DEHYDRATION
Following the eosin stain, the slide is
passed through several changes of
alcohol to remove all traces of water.
STEP 7: DEHYDRATION
While staining, water can get into the
alcohols (following eosin) due to
carryover between steps. When reagents
are not changed regularly, the water
content will continue to increase and will
be transferred to the xylenes that follow
before cover-slipping.
This excess water in the xylene can, over
time, cause seeping of the eosin from the
tissue. It is seen on the slide as a pink
haze.
This artifact can occur even if the xylene
is not visibly contaminated with water.
FIGURE: The water in this section has
removed nearly all of the cytoplasmic
counterstain. Note the water droplets
surrounding the section.
STEP 8: CLEARING
The slide is then rinsed in several baths
of xylene which “clears” the tissue and
renders it completely transparent.
A thin layer of polystyrene mountant is
applied, followed by a glass coverslip. If
the stain and all the subsequent steps
have been properly performed, the slide
will reveal all the important microscopic
components and be stable for many
years.
MOUNTING
In order to create permanent slides for
histopathology, mounting media are
needed. They hold the specimens in
place, in between the slide and cover slip,
and allow the specimen to be stored for
years. Mounting media also provide the
maximum degree of transparency to
stained tissue sections.
The refractive index of the mounting
medium must be close as possible to that
of the tissue (1.52 - 1.54) to make the
tissue section more transparent.
MOUNTING
The typical mounting media used for
routine histopathology specimen is
Canada balsam.
The mounting medium is a resin that
hardens in small amounts. As they are
placed between the coverslip and slide, it
will harden.
MOUNTING
Care must be taken to ensure that
bubbles do not appear in between the
tissue and the cover slip. This is because
bubbles have a tendency to retract and
interfere with the microscopic evaluation
of tissue sections.
The preparation should also be done
neatly. If bubbles appear, the slides are
then returned to xylene to remove the
cover slip, and the slide is then
remounted.

MOUNTING
Bubbles during mounting.

MOUNTING
Glass slides should not be allowed to dry
before applying a cover slip. Air drying
produces a light brown stippling on the
surface of the specimen which resembles
a pigment.

MOUNTING
The mounting medium must be of the
appropriate viscosity.
Decreased transparency results from a
medium that is too viscous.
Retraction of mounting medium also
occurs when it is diluted with too much
solvent

END