troubleshooting fixation

Question 1

autolysis cause by

Answer 1

caused by delayed fixation

Question 2

loss of nuclear chromatin
loss of cells
shrinkage and artificial space

prevention :

Answer 2

not preventable on autopsy tissue

place specimen in fixative ASAP

open and pin sections

slice large pieces into smaller

Question 3

incomplete fixation appearance and prevention

Answer 3

tissue may separate on flotation bath
poor tissue morphology
smudgy nuclei with no chromatin pattern **
enter of tissue more eosinophilic due to alcohol fixation

prevention

• longer in fixative
• change to a faster fixative
• formalin alcohol on first 3 stages of processing
• ensure gross specimens are thin enough
• dont pack cassettes
• increase temp to 37 degrees

Question 4

secondary / post fixation reason

Answer 4

section cuts easier

staining is more accurate

after gluteraldehyde fiction for EM, osmium may further stabilize the tissue

aka post mordanting

Question 5

what is NBF the universal fixative

Answer 5

using one fictive allows for fiction ASAP in OR or dr offices

NBF allows for wider variety of stains than any other
- compatible with most stains

Question 6

how do you reverse fixation

Answer 6

YOU CANT. DONT FUCK IT UP

Question 7

Best fixative for lipids

Answer 7

osmium tetroxide- and turn black

lipids are lost during processing gif NBF is used

generally frozen sections are used for this purposed

Question 8

carbohydrates fixative

Answer 8

no single ideal fixative

generally alcohol based
- prevents glycogen streaming

Question 9

acid fast bacilli; avoid

Answer 9

avoid alcohol fixation as it leads to false negative staining reactions

Question 10

urates ( uric acid) not soluble in

Answer 10

not soluble in alcohol or ether
absolute alcohol or carney could be used
usually frozen sections are used rather than fixation
- demonstrates uric crystals

Question 11

immunofluorescence or immunoperoxidase may be performed on tissue fixed with

Answer 11

may be performed on NBF fixed

depends on antigen-antibody stability
frozen sections receive a brief methanol or acetone fixation

Question 12

enzyme histochemistry

Answer 12

enzymes are destroyed by fixation

muscle biopsies are flash frozen

cut on cryostat(-20) stored at -70degrees ( ultraslow freezer)

Question 13

electron microscopy

Answer 13

fixation must be EXCELLENT - higher magnification and resolution is not as forgiving

ideal tissue is 0.5-1mm cube ( most failures involve large tissue )

gluteraldehyde in fridge for 2 hrs followed by buffer or post fixation ( osmium )