PAP STAINING PROTOCOL

REAGENT: 96° ALCOHOL (3 MINUTES)

Function: Initial fixative, dehydrates and fixes the sample to the slide. It can also remove impurities before staining.

STEP 1

STEP 2

REAGENT: COMMON WATER (15 DIPS)

FUNCTION: It is used to hydrate the sample after the alcohol and before the hematoxylin.

(IT IS RECOMMENDED TO DO THIS STEP 2 OR 3 TIMES)

STEP 3

REAGENT: HARRIS HEMATOXYLIN (7 TO 9 MINUTES)

FUNCTION: Basic nuclear stain: stains cell nuclei dark blue/black, allowing shape and chromatin to be observed.

A circular microscopic view showing a tissue sample, characterized by a complex pattern of cell structures in various shades of purple and pink against a dark background.

STEP 5

REAGENT: ACID ALCOHOL (1 DIP)

FUNCTION: It acts as a differentiator, removing excess hematoxylin to leave only the dye well-adhered to the nucleus. It improves contrast.

STEP 6

REAGENT: COMMON WATER (15 DIPS)

FUNCTION: It is used to hydrate the sample after the alcohol and before the hematoxylin.

(IT IS RECOMMENDED TO DO THIS STEP 3 TIMES)

STEP 7

REAGENT: LITHIUM CARBONATE (30 DIPS)

FUNCTION: Neutralizes the acid in the acidic alcohol and changes the hue of the nucleus from red to blue (nuclear enhancement). This is a key step in improving contrast.

STEP 9

REAGENT: 96° ALCOHOL (15 DIPS)

FUNCTION: THE SAMPLE MUST BE DEHYDRATED TO ENTER THE ORANGE, SINCE THIS IS AN ALCOHOLIC BASE. SKIPPING THIS STEP COULD CAUSE THE DYE NOT TO ADHER WELL TO THE CELLS.

STEP 10

REAGENT: ORANGE G ( 2 TO 3 MINUTES )

FUNCTION: It stains keratinized cells (such as superficial squamous cells) and KERATIN orange.

STEP 11

REAGENT: 96° ALCOHOL (15 DIPS)

FUNCTION: THE SAMPLE MUST BE DEHYDRATED TO ENTER THE EA-50, SINCE THIS IS AN ALCOHOLIC BASE. SKIPPING THIS STEP COULD CAUSE THE DYE NOT TO ADHER WELL TO THE CELLS.

STEP 12

REAGENT: EA-50 ( 2 TO 4 MINUTES )

FUNCTION: Mixed cytoplasmic staining: stains non-keratinized cytoplasm. Eosin → pink. Green/blue → turquoise to green hues depending on intracellular pH.

STEP 13

REAGENT: 96° ALCOHOL (REPEAT 3 TIMES) (15 DIPS)

Function: DEHYDRATE SAMPLE AND CLEAN EXCESS OF OLD DYES.

STEP 14

REAGENT: ABSOLUTE ALCOHOL (15 DIPS)

Function: Final dehydration completely removes water before mounting with xylene. Increases cell transparency.

STEP 15

REAGENT: XYLOL ALCOHOL (15 DIPS)

Function: A blend that aids the transition between alcohol and pure xylene. It improves penetration and prevents the formation of droplets or bubbles.

STEP 16

REAGENT: XYLOL (15 DIPS)

Function: Clearing and mounting, cleans and clears the sample. It is immiscible with water, so it is used after dehydration. Leaves the slide ready for permanent mounting.