PRESSBOOK

● Structures of cells and tissues can be distinguished at two levels

1. Fine structure: distinguished at the level of compound light microscopy (1000x magnification or
less)
2. Ultrastructure: detailed structure of the cell cytoplasm, organells and membranes. Studied with
electron microscope

LAB

STAINING LAB OUTLINE

● The process through which cell structure is preserved is called fixation

● Cell structure is most commonly studied in slices of the tissue, called sections, that are thin
enough to allow transmission of light or an electron beam
○ Paraffin method used to section tissues

Paraffin method steps (ones we did in lab highlighted):

1. Tissue resection (removing the tissue of interest from the organism)

● Preserve the cellular structure of the tissue

1. Fixation (halting cells in their current state)

● skin tissue is dehydrated through a series of graded ethanol baths to displace the water, and
then infiltrated with wax
● Isopropyl alcohol (IPA) is a favored reagent because it is miscible in paraffin

1. Dehydration
2. Infiltration and embedding (for the paraffin method, the tissue is embedded in paraffin)
● Infiltration:
○ Prior to sectioning, the tissue block must be infiltrated with a material that acts as a
support during the sectioning process
○ The paraffin sectioning method is the most widely used method for performing
histological analysis
○ During infiltration, the paraffin will equilibrate within the tissue, eventually occupying all
of the space in the tissue that was originally held by IPA
● Embedding: the tissue is allowed to solidify in a mold, and embedded within a small cube of
paraffin

1. Sectioning with a microtome

● Sectioning is accomplished by using a cutting apparatus called a microtome
● The microtome will drive a knife across the surface of the paraffin cube and produce a series of
thin sections of very precise thickness

1. Mounting sections on microscope slides

● Sections are carefully transferred into a water bath held at 45◦C
● Sections flatten and the wrinkles disappear
● A clean microscope slide is dipped into the bath, and slowly pulled upward, out of the solution,
allowing sections to adhere to the surface
● Slide is oriented with the label facing upward

1. Clearing and staining

● Before a section can be stained, the paraffin must be removed, a process called clearing
● After clearing, only the tissue remains adhered to the slide
● Clearing is accomplished by passing the mounted sections through the solvent xylene, which
dissolves the paraffin
● Staining of histological sections allows observation of features otherwise not distinguishable
● For routine histological work, it is customary to use two dyes, one that stains certain
components a bright color and the other, called the counterstain, that stains other cellular
structures a contrasting color\
● 2 stains most widely used for routine work are hematoxylin and eosin (H&E)
● Hematoxylin stains negatively charged structures, such as DNA, a blue color
● Eosin imparts a pink color to most of the other cell components.

1. Preparation of permanent mounts

● Permanently mount the sections under a coverslip
● Accomplished by covering the section in a medium that will harden and produce a clear binder
between the slide and cover slip
● The ideal mounting medium should not distort the stain colour, or yellow and become brittle
with age
● Used a mounting resin called Permount

Lab Part 1: Staining and Coverslipping

● Stain a slide and then add a coverslip to prepare a permanent mount.

● After staining, each student will attempt to place a coverslip over the stained tissue

Materials
● Staining dishes
● Vertical slide holders: Used to hold slides and transfer them between solutions
● Coverslipping materials

Lab Protocol 1: Clearing and Staining Tissue

 3 main parts:
1. Clearing and rehydrating (steps 1-10 of the protocol)
2. Staining & rinsing (steps 11-20 of the protocol)
3. Dehydrating (steps 21-30 of the protocol)

Lab Protocol 2: Preparing permanently mounted sections

● Remove excess xylene from around the tissue
● Place a thin line of resin solution (=Permount) along the bottom edge of the slide.

MICROSCOPY LAB OUTLINE

● Use the compound light microscope to capture images from slides containing prepared mouse
skin cross sections
● You will then analyze the images and evaluate if there are changes in the integumentary (skin)
structure associated with age
● The ages of the mouse tissues when they were resected and fixed are:
○ Mouse: 5-day
○ Mouse: 30-day
○ Mouse: 2-year

1. The sagittal plane sections the body into left and right sides
2. The frontal plane sections the body into anterior and posterior regions
3. The transverse plane sections the body into superior and inferior regions

When using the paraffin method, the tissue is sliced by the microtome parallel to the base of the
mold used to embed the tissue

In this lab:
1. Students will practice embedding a piece of pseudotissue
2. Students will capture images from the prepared slides of mouse skin sections using the
compound microscope.
Materials
1. Embedding station (see figure 8)
2. Prepared slides of mouse skin tissue:
3. Mouse: 5-day
4. Mouse: 30-day
5. Mouse: 2-year
6. Compound Light Microscope
7. ImageJ

Lab Protocol 3: Embedding

● orient the tissue as if you were creating a longitudinal section

1. Place the mold under the wax dispenser, 2-3 mm of paraffin around all edges as a buffer zone
when sectioning.
2. Snap off the top of the cassette and set aside.
3. Fill the mold 1/4 of the way by pushing the wax dispenser with your forceps or finger.
4. With the forceps provided, carefully transfer your pseudotissue into the mold and orient it as
needed for a cross section or longitudinal section
5. To keep the pseudotissue in the desired orientation, slide the mold over to the cold spot
6. Take the cassette and place it on the mold
7. Push it in gently with forceps to keep it in place and fill with wax almost completely.
8. Slide it to the cooling place on the right
9. Wait 15-20 minutes before pulling the cassette from the mold. Return the mold to its
compartment at E

Lab Protocol 4: Image Capture and Analysis

1. Use the Zeiss compound light microscope to capture 4-5 images at different locations (=regions
of interest; ROIs) from each slide. Use the 10X magnification objective (100X total magnification)
2. Make sure you capture images of the stage micrometer at the same magnification and with the
same microscope you used to capture your images of the skin tissue
3. For image analysis, measure the thickness of the epidermis and dermis in each ROI for the 5-day,
30-day, and 2-year mouse images, then calculate the mean thickness of the dermis and
epidermis for these samples. (Note: we suggest you take 10 measurements of each tissue layer
for each age of mouse tissue – these 10 measurements can be spread among the 4-5 ROI images
for each age at your discretion).