Ultrashort Cycle 1 hour 41 minutes (includes fixation time)

STEP

REAGENT

TEMPERATURE (°C)

VACUUM

IMMERSION

1

100% ethanol

45

Y

5 minutes

2

100% ethanol

45

Y

5 minutes

3

100% ethanol

45

Y

5 minutes

4

100% ethanol

45

Y

10 minutes

5

chloroform

45

y

10 minutes

6

chloroform

45

y

10 minutes

7

chloroform

45

Y

10 minutes

8

wax

62

Y

10 minutes

9

wax

62

Y

10 minutes

Short Cycle 3 hour 31 minutes (includes fixation time)

STEP

REAGENT

TEMPERATURE (°C)

VACUUM

IMMERSION

1

100% ethanol

45

Y

20 minutes

2

100% ethanol

45

Y

10 minutes

3

100% ethanol

45

Y

10 minutes

4

100% ethanol

45

Y

15 minutes

5

100% ethanol

45

Y

20 minutes

6

chloroform

45

N

20 minutes

7

chloroform

45

N

10 minutes

8

chloroform

45

N

20 minutes

9

wax

63

Y

30 minutes

10

wax

63

Y

20 minutes

Overnight Cycle 11 hours 36 minutes (includes fixation time)

STEP

REAGENT

TEMPERATURE (°C)

VACUUM

IMMERSION

1

70% ethanol

45

Y

30 minutes

2

100% ethanol

37

Y

1 hours

3

100% ethanol

37

Y

20 minutes

4

100% ethanol

37

Y

20 minutes

5

100% ethanol

37

Y

20 minutes

6

100% ethanol

37

Y

30 minutes

7

100% ethanol

37

Y

1 hours

8

chloroform

37

N

1 hours

9

chloroform

37

N

1 hours

10

chloroform

37

N

1 hours

11

wax

60

Y

2 hours 30 minutes

12

wax

60

Y

1 hours 30 minutes

'Jumbo' Cycle 16 hours 36 minutes (includes fixation time)

STEP

REAGENT

TEMPERATURE (°c)

VACUUM

IMMERSION

1

70% ethanol

37

Y

1 hours

2

100% ethanol

37

Y

1 hours

3

100% ethanol

37

Y

1 hours

4

100% ethanol

37

Y

30 minutes

5

100% ethanol

37

Y

30 minutes

6

100% ethanol

37

Y

1 hours

7

100% ethanol

37

Y

1 hours

8

chloroform

37

N

2 hours

9

chloroform

37

N

1 hours 30 minutes

10

chloroform

37

N

1 hours 30 minutes

11

wax

62

Y

2 hours 30 minutes

12

wax

62

Y

2 hours 30 minutes

Table 6.1. Processing Cycles for Microwave-Fixed Tissues

All tissue blocks are microwave irradiated in batches in normal saline to a solution temperature of 62°C and processed according to the following cycles in an automated tissue processor. The times indicated do not include the times required for the changing of reagents, this varying with the tissue processor employed.

Ultrashort cycles (1 hour 41 minutes) can be applied to small specimens such as endoscopic and needle biopsies, whereas the short cycle (3 hour 31 minutes) can be used for virtually all other tissue blocks except for those composed of dense connective tissue such as myometrium and skin or those with a high content of lipid. Such specimens require a short period of immersion, such as 10 minutes, in 10% buffered formalin before irradiation in normal saline and lipid-rich tissues should, in addition, be briefly cleared in 70% alcohol before exposure to microwaves.

Some Common Fixatives

K Values

4% formaldehyde

0.78

100% ethanol

1.0

1.2-4.0% glutaraldehyde

  

- at 4°C

0.33-0.5

- at room temperature

0.5-1.0

5% acetic acid

1.2

0.5% chromium trioxide

0.25

0.5-2.0% osmium tetroxide

0.29-0.58

saturated picric acid

0.5

3% potassium dichromate

1.33

saturated mercuric chloride

0.78-0.84

Table 6.2. K Values for some Common Fixatives