HISTO LAB - CLEARING & INFILTRATION

DEHYDRATION

This process is commonly carried out by immersing specimens in a series of ethanol (alcohol) solutions with increasing concentration to avoid excessive distortion of tissue until a water-free tissue in alcohol is reached.

Water soluble proteins

are removed at lower concentrations of ethanol. When ethanol concentration is increased to 100%, certain lipids may be dissolve

70% ethanol 15 min 90% ethanol 15 min 100% ethanol 15 min 100% ethanol 15 min 100% ethanol 30 min 100% ethanol 45 min

TYPICAL dehydration sequence for specimens not more than 4mm thick would be

CLEARING

The dehydrated tissue is transferred to an intermediate solvent that is fully miscible with both ethanol and paraffin wax.

Ethanol and paraffin wax.

CLEARING is when the dehydrated tissue is transferred to an intermediate solvent that is fully miscible with both __________ and _________

optical clarity or transparency

The term “clearing” has been chosen because many (but not all) clearing agents impart an _________ to the tissue due to their relatively high refractive index.

high refractive index

The term “clearing” has been chosen because many (but not all) clearing agents impart an optical clarity or transparency to the tissue due to their relatively ________

fat

Another important role of the clearing agent is to remove a substantial amount of ________ from the tissue which otherwise presents a barrier to wax infiltration.

Xylene 20 min, Xylene 20 min, Xylene 45 min

A typical clearing sequence for specimens not more than 4mm thick would be

INFILTRATION

The cleared tissue is infiltrated with a suitable histological wax (usually paraffin) which is liquid at 60°C and then allowed to cool to 20°C in order to solidify into a consistency that allows sections to be cut.

60°C

The cleared tissue is infiltrated with a suitable histological wax (usually paraffin) which is liquid at ________ and then allowed to cool to 20°C in order to solidify into a consistency that allows sections to be cut.

20°C

The cleared tissue is infiltrated with a suitable histological wax (usually paraffin) which is liquid at 60°C and then allowed to cool to __________in order to solidify into a consistency that allows sections to be cut.

Histological wax

These waxes are mixtures of purified paraffin wax and various additives that may include resins such as styrene or polyethylene that allow them to be sectioned thin enough on a microtome, forming ribbons that can flatten fully when floated on a warm water bath

styrene or polyethylene

These waxes are mixtures of purified paraffin wax and various additives that may include resins such as _____or _______ that allow them to be sectioned thin enough on a microtome, forming ribbons that can flatten fully when floated on a warm water bath

Paraffin wax 30 min, Paraffin wax 30 min, Paraffin wax 45 min

A TYPICAL PARAFFIN INFILTRATION sequence of paraffin for specimens not more than 4mm thick would be

DEHYDRATION

This process of removing intercellular water from the tissue following fixation and prior to wax impregnation is known as

CLEARING

is the process whereby alcohol is removed from the tissues and replaced with a substance that will dissolve the wax with which the tissue is to be impregnated.

IMPREGNATION

is the process whereby the clearing agent is completely removed from the tissues and replaced by a medium that will completely fill all the tissue cavities, thereby giving a firm consistency to the specimen, and allowing easier handling and cutting of suitably thin sections without any damage or distortion to the tissue and its cellular components

Forceps, Specimen Label, Tissue Capsule, 95% Alcohol, Chloroform, Tissue Basket, Pencil, 100% Alcohol, Melted Paraplast, 80% Alcohol

EQUIPMENT AND MATERIALS USED IN CLEARING AND INFILTRATION

Formalin-fixed tissue

SPECIMEN

6. Dehydrate in 80% alcohol for 1 hour

7. Dehydrate in 95% alcohol for 1 hour

8. Dehydrate in 95% alcohol for 2 hours (2nd change)

9. Dehydrate in 100% alcohol for 2 hours

10. Dehydrate in 100% alcohol for 2 hours (2nd change) or overnight.

11. Clear in chloroform for 1 hour

12. Clear in chloroform for 1 hour (2nd change).

13. Impregnate with melted paraffin for 2 hours.

14. Impregnate with melted paraffin for 2 hours

15. Impregnate with melted paraffin for 2 hours, solidify overnight

16. Warm paraffin until melted before embedding.

DEHYDRATION procedure in laboratory

60%

Dehydration is best accomplished with the use of a series of progressively increasing concentrations of alcohol beginning with _____

shrinkage and hardening

Under no circumstances should a formalin-fixed tissue be transferred directly to higher grades of alcohol, e.g. 85-95% alcohol because this is liable to produce considerable ______________ of tissues leading to distortion. The word clearing is used because in addition to removing alcohol, many of these have the property of making tissues transparent due to their high index of refraction

Immiscible

After complete dehydration, tissues must undergo clearing before paraffin embedding, as ethanol and paraffin wax are ______

proper structural integrity and staining quality

The dehydration process is an essential histological step that prepares tissues for embedding, ensuring ________ and _______

Gradual ethanol immersion

minimizes tissue shrinkage and artifacts, while additional steps such as solvent substitution for fatty tissues and temperature control enhance processing efficiency. Proper dehydration ultimately ensures high-quality microscopic examination.

Hydrophobic

Since paraffin is_______ , any residual water prevents proper infiltration, leading to poor sectioning and staining

graded ethanol series

The process follows a ___________ to prevent tissue distortion and shrinkage.

acetone and isopropanol

Alternative dehydrants such as _________ and ________are used for fatty tissues that ethanol alone cannot process efficiently.