Histology Lab

3 anatomical planes in humans

sagittal, frontal, transverse

Sagittal plane

Sections the body into left and right sides

Frontal plane

Sections the body into anterior (front) and posterior (back) regions

Transverse plane

Sections body into superior (top) and inferior (bottom) regions

Sagittal plane

Frontal plane

Transverse plane

Tissue biopsy

Minor surgical procedure that cuts away a piece of tissue from the organism being studied

Paraffin method

Tissue is sliced by the microtome parallel to the base of the mold used to embed the tissue

Sagittal and frontal planes section a human similarly to the __________ section of a nematode

Longitudinal

Embedding and orienting tissue for transverse sections

Longitudinal axis of the tissue is perpendicular to the base of the mold

Embedding and orienting tissue for longitudinal sections

Longitudinal axis of the tissue is parallel to the base of the mold

How to orient and embed for a transverse section

How to orient and embed for a longitudinal section

Embedding and Orienting Tissue Steps

1. Place tissue in mold with the longitudinal axis either parallel (for longitudinal section) or perpendicular (for transverse section) to the mold base

2. Pour liquid paraffin into the mold

3. Place cassette on top of the mold

4. Allow paraffin to cool and solidify

5. Remove cassette from mold

What is the tissue attached to after the paraffin solidifies

The cassette

Embedding and Orienting Step 1

Place tissue in mold with the longitudinal axis either parallel (for longitudinal section) or perpendicular (for transverse section) to the mold base

Embedding and Orienting Step 2

Pour liquid paraffin into the mold

Embedding and Orienting Step 3

Place cassette on top of the mold

Embedding and Orienting Step 4

Allow paraffin to cool and solidify

Embedding and Orienting Step 5

Remove cassette from mold

Cutting a Tissue with Microtome Steps

1. Attach cassette with the tissue to the block holder in the microtome

2. Turn the crank wheel to move the block holder up and down against the stationary blade

3. Transfer the tissues to a warm water bath

4. Stain the tissue

The Paraffin Method Steps

1. Tissue resection - removing the tissue of interest from the organism

2. Fixation - Halting cells in their current state

3. Dehydration - Series of alcohol baths dehydrates the tissue

4. Infiltration and embedding - Infiltrated with a material that acts as a support during sectioning

5. Sectioning - Using microtome

6. Mounting - Adhering the slices onto microscope slides

7. Cleaning and staining - Rehydration and staining of the cellular structures

8. Preparation of permanent mounts - Using resin to adhere the coverslip

Paraffin method step 1

Tissue resection - removing the tissue of interest from the organism

Paraffin method step 2

Fixation - Halting cells in their current state

Paraffin method step 3

Dehydration - Using a series of alcohol baths

Paraffin method step 4

Infiltration and embedding - Material is infiltrated and acts as a support for sectioning

Paraffin method step 5

Sectioning - Using microtome

Paraffin method step 6

Mounting - Adhering slices to microscope slides

Paraffin method step 7

Clearing and staining - Rehydrating and staining cellular structures

Paraffin method step 8

Preparation of permanent mounts - Using resin to adhere coverslip

How many steps are in clearing and staining tissue

23

3 main parts of clearing and staining tissue

1. Clearing and rehydrating (steps 1-9)

2. Staining and rinsing (steps 10-17)

3. Dehydrating (steps 18-23)

Clearing and rehydrating steps

1. Xylene I (dip 10x, leave 3 mins)

2. Xylene II (dip 10x, leave 3 mins)

3. Xylene III (dip 10x, leave 3 mins)

4. 100% ethanol I (dip 10x, leave 1 min)

5. 100% ethanol II (dip 10x, leave 1 min)

6. 100% ethanol III (dip 10x, leave 1 min)

7. 95% ethanol (dip 10x, leave 1 min)

8. 70% ethanol (dip 10x, leave 1 min)

9. Running water (2 min)

Clearing and rehydrating reagents used

• Xylene I, II, III (dip 10x, leave 3 min)

• 100% ethanol I, II, III (dip 10x, leave 1 min)

• 95% ethanol (dip 10x, leave 1min)

• 70% ethanol (dip 10x, leave 1 min)

• Running water (leave 2 min)

Staining and rinsing steps

1. Modified Mayer’s Hematoxylin (dip 10x, leave 5 mins)

2. Running water (leave 1 min)

3. Weak acid alcohol (dip 10x)

4. Running water (leave 1 min)

5. Bluing agent (dip 10x, leave 1 min)

6. Running water (leave 1 min)

7. 95% ethanol (dip 10x)

8. Alcoholic 0.5 eosin pH5.5 (dip 10x, leave 30 seconds)

Dehydrating steps

1. 95% ethanol I (dip 10x, leave 1 min)

2. 100% ethanol I (dip 10x, leave 1 min)

3. 100% ethanol II (dip 10x, leave 1 min)

4. Xylene I (dip 10x, leave 2 min)

5. Xylene II (dip 10x, leave 1 min)

Where to put slides when waiting to use them

Leave them in the xylene

Resin solution name

Permount

How long should slides air dry for the resin to set

At least 24 hours

The 2 skin layers we measured

Epidermis and dermis

Age of mouse samples

5 day and 2 year