Immunohistochemistry

Routine Paraffin Cytoplasmic or Membrane Antigens.

Nuclear Antigens

FIXATION AND PROCESSING OF MOUSE TISSUES

Our laboratory has been experimenting with the fixation and processing mouse tissue for Immunohistochemistry and In Situ Hybridization. The following recommendations come from this experience.

GENERAL:
1) No ONE fixative works for all antigens and all antibodies. Conditions must be optimized for each antigen-antibody combination. When first starting, we recommend that you place the tissue in a variety of fixatives to find the optimum for your tissue, antigen and antibody.
2) Fixation should be rapid but thorough.
a) The optimal tissue size is no thicker than 1-2mm. If a tissue is thicker, the investigator should fix for one hour, and then cut 1mm thick sections ("bread-loaf") so that all surfaces are exposed equally.
b) The Volume of fixative should be at least 10x the volume of the tissue. If the tissue is "bloody", remove the bloody fixative after an hour and add fresh fixative.
c) A tissue can be placed in one fix and then portions placed in a second fixative for "mordanting". (Example: fix in paraformaldehyde for ISH. Place a paraformaldehyde sample in Bouin's Fix for IHC.)
3) Once the tissue is fixed, it should be held in 70% alcohol and processed promptly.
4) Paraformaldehyde is the best fixative for ISH. Bouin's Fixative is the best all-around fixative for IHC. (Do not use the Bouin's substitute without Picric Acid. The Picric Acid is required for best results.) However, excellent results have been obtained with Carnoy's, B5, and formaldehyde. Zenker's and Telly's fixatives have seldom provided satisfactory results.

PROTOCOL:
1) Remove tissue as rapidly as possible. Place in 10 volumes of fixative. Cold fixative is better than warm or room temperature fixative.
2) After one hour, inspect sample. If the fixative is cloudy, replace with fresh fixative. If the sample is thick, remove sample, place on a cutting board, cut 1-2mm slices to expose all surfaces and return to 10 volumes of fixative. (note: the short fixation will firm up hard-to-slice tissues such as lymph nodes, spleen and thymus, making them easier to slice).
3) Fix for six to 12 hours of over night BUT no longer. Remove and place in 70% ETOH until shipping or processing.

SAMPLING:
1) If the tissue is of interest, make sure that at least 50% of the sample is composed of normal adjacent tissue.
2) In the case of the mammary gland, the gland should be spread on a slide so that the fat does not inhibit fixation. After an hour, the flattened mammary gland can be removed from the slide and placed in more fixative.
3) If one organ is diseased, make sure that the contralateral organ is sampled (if the organs are bilateral).
4) The best interpretation is provided when age matched control organs are provided.

INFORMATION:
1) Make sure that the sample is submitted with the appropriate request forms that includes demographic and experimental data about the animal.

Paraffin

1.  Xylene	                  5 min.
2.  Xylene	                  5 min.
3.  Xylene/Lugol’s	          4 min. *
4.  Absolute ETOH	          3 min.
5.  Absolute ETOH	          3 min.
6.  MEOH/H2O2(0.3%)	         20 min.
7.  Absolute ETOH	          1 min.
8.  95% ETOH	                  2 min.
9.  70% ETOH	                  2 min.
10. H2O (running)	          2 min.
11. Dist. H2O	                  2 min.
12. PBS (x2)	                  5 min.
13. Normal Equine Serum 10%	 20 min.
14. Primary abs. (Dilutions vary)   Overnight
15. PBS (x2)	                   5 min.
16. Equine Anti-Mouse Biotin 
                    Conjugate	  60 min.
17. PBS (x2)	                   5 min.
18. ABC - Elite	                  30 min.
19. PBS (x2)	                   5 min.
20. DAB (Vector-Brown)	         3-5 min.
22. Mayer’s Hematoxylin	           1 min.
23. Tap H2O	                5-10 min.
24. Dehydrate, clear and coverslip	

* B5 Fixed Tissue only

NOTE:  Tissue sections are placed in a 55 Celsius oven for 30 minutes or overnight.

Immunoperoxidase Staining (Nuclear Antigens)

1.  Xylene	                   5 min.	 RoomTemp.
2.  Xylene	                   5 min.	 RoomTemp.
3.  ETOH	                   3 min.	 RoomTemp.
4.  ETOH	                   3 min.	 RoomTemp.
5.  H202 + 3% Methanol	          20 min.	 RoomTemp..
6.  ETOH	                   2 min.	 RoomTemp.
7.  ETOH	                   2 min.	 RoomTemp..
8.  H2O tap	                   5 min.	 RoomTemp..
9.  distilled H2O	           5 min.	 RoomTemp..
10. Citrate Buffer	           4 min.	microwave*
11. Citrate Buffer	           4 min.	microwave*
12. Citrate Buffer	           4 min.	microwave
13. Cool down	                  15 min.	 RoomTemp.
14. PBS	5 min.	                                 RoomTemp..
15. PBS	5 min.	                                 RoomTemp.
16. N horse serum	          20 min.	 RoomTemp.
17. Primary antibody            overnight           4 C 
18. PBS	5 min.	                                 RoomTemp.
19. PBS	5 min.	                                 RoomTemp.
20. Secondary (BHAM) 1:800	  60 min.	 RoomTemp.
21. PBS	5 min.	                                 RoomTemp.
22. PBS	5 min.	                                 RoomTemp.
23. Tertiary ABC 1:50	          30 min.	 RoomTemp.
24. PBS	5 min.	                                 RoomTemp.
25. PBS	5 min.	                                 RoomTemp.
26. DAB	3-5 min.	                         RoomTemp.
27. H2O running tap	           5 min.	 RoomTemp.
28. Hematoxylin-Mayers	          30 seconds	 RoomTemp.
29. H2O running tap		
30. Dehydrate, clear, coverslip		

Center for Comparative Medicine
University of California, Davis, CA 95616
Phone: 530-752-5524
Fax: 530-752-7914
EMail: jewalls@ucdavis.edu

LAST UPDATED April 24, 2000