Papanicolaou Staining: A Technique to Visualize the Cytological Features of Tubal Cells

Protocol

1. Modified Papanicolaou Staining Protocol

1. Stain the tissue smeared slides by running the automatized non-gyn stain procedure, as illustrated in Table 1.
   NOTE: The staining method represents a modification of the Papanicolaou staining technique that we use routinely for non-gynecologic specimens. We use an automated tissue processor to stain some slides. We chose the Non-Gyn staining procedure over the Gyn staining procedure in this study. The Eosin used is EA-65, instead of the EA-50 used for gynecological specimens (Pap smear specimen). In addition, OG-6 in the Gyn staining procedure, which is mainly for squamous cell staining, is not included in the staining procedure since no squamous cells are expected to be seen in this study. EA65 (Eosin Azure) is a counter stain solution comprised of 3 dyes: Eosin Y, Fast green FCF, and Bismarck brown Y. The procedure is illustrated in Table 1.
2. Perform a manual quick Pap stain procedure as follows:
   1. Dip the tissue smeared slide into 95% ethanol, 10 times, with each dip taking about 1 s.
   2. Dip the slide into H₂O, 10 times, with each dip taking about 1 s.
   3. Emerge the slide in hematoxylin for 10 - 60 s.
   4. Dip the slide into H₂O, 10 times, with each dip taking about 1 s.
   5. Dip the slide into 95% ethanol,10 times, with each dip taking about 1 s.
   6. Emerge the slide in EA65 for 1 - 3 min.
   7. Dip the slide into H₂O, 10 times, with each dip taking about 1 s.
   8. Dip the slide into 95% ethanol, 10 times, with each dip taking about 1 s.
   9. Dip the slide in xylene until the slide becomes clear.
      NOTE: This is a modified Papanicolaou staining procedure. This method is used as a more rapid stain method for cases with limited time or space like on-site fine needle aspiration and STAT specimens. This procedure provides comparable quality staining as the automatized Non-Gyn stain procedure. The procedure is illustrated in Table 2.

Table 1: Non-Gyn Stain Steps.

95% Ethanol	5 min
H2O	10 sec
Hematoxylin	15 sec - 3 min
Distilled H2O	5 min
95% Ethanol	30 sec
EA65	2 - 5 min
95% Ethanol	30 sec
95% Ethanol	30 sec
100% Ethanol	30 sec
100% Ethanol	30 sec
Xylene	30 sec
Xylene	5 min

Table 2: Manual Quick Pap Stain Steps.

95% Ethanol	10 dips
H2O	10 dips
Hematoxylin	10 sec-1 min
Distilled H2O	10 dips
95% Ethanol	10 dips
EA65	1 - 3 min
95% Ethanol	10 dips
100% Ethanol	10 dips
Xylene	Dip until clear

Materials

Name	Company	Catalog Number	Comments
Papanicolaou Stain, EA 65	sigma aldrich	HT40432
95% Ethanol	sigma aldrich	652261
100% Ethanol	sigma aldrich	493538
Xylene	sigma aldrich	214736
Hematoxylin	sigma aldrich	H9627