Re: [Histonet] Unstained slides precut for IHC

[Tony Henwood (SCHN) via Histonet]

Hi Carrie,
Over-heating of sections (and tissue blocks) for IPX is probably one of the 
most significant and unappreciated  pre-analytical factors that can affect 
immunolocalisation. We closely scrutinise slides closely for overheating, 
especially those sent to us for immunostaining. We strongly prefer air-dried 
sections that we place on our immunostaniners (we have both a Bond and Ventana 
Benchmark). The following might be useful:

Controlled Section Baking for Immunohistochemistry
One source of poor immunostaining is overheating of tissue and sections. 
Several authors have reported that heated slide drying adversely affects 
sensitivity in immunohistochemistry (1). Therefore, some have advocated the use 
of lower temperature drying using adhesive-coated slides to improve the 
sensitivity of the test (2-5).
In one study (1), half the antigens were adversely affected by section drying 
at 80oC including 5D3, CMV, S100, HMB45 and CEA. Oates (4) used antisera to 
epithelial membrane antigen from three different companies and found that for 
slides dried at 58"C, staining was often paler than slides dried at room 
temperature or at 37°C.
Low heat attachment of sections to slides can cause several issues including 
inadequate attachment of the tissue sections so that tissue sections may be 
lost during antigen recovery and/or immunostaining, the inability of some 
paraffins to melt well at 58oC, and the requirement of more than 1 hr before an 
immunohistochemical procedure may be started. It has been recommended that the 
most efficient protocol for mounting tissue sections to microscopic slides 
would be to attach the tissues overnight before applying the 
immunohistochemical procedure at a temperature at which all tissue mounting 
paraffins should melt (e.g., 65oC) (6). It should be remembered that a 
significant dewaxing of sections occurs when slides are heated a few degrees 
above the melting point of the wax. 
Laboratory ovens seem to be variable in their ability to maintain a constant 
temperature with the implication that it is possible to either over-cook 
sections thus adversely affecting antigens or under-heat them, possibly 
compromising subsequent de-waxing. There is also the human element. How often 
are slides removed from the oven at the required time?
The modern automatic immunostainers have excellent on-board slide heating to 
achieve reproducible, accurate antigen retrieval. This feature also allows 
controlled “baking” of sections and being able to programme a set time, removes 
the possibility of human error. At the Children’s Hospital, the Bond 3 is used 
for automated immunohistochemistry. A study was designed to assess the 
usefulness of on-board baking in routine immunohistochemistry.
Control sections were immunostained for several antigens (see table) using the 
Bond 3 on-board baking and dewax facility. Freshly cut sections were dried at 
37oC for 5 minutes to remove excess water. Slides were then loaded onto the 
Bond and the baking procedure used was 35 minutes at 63oC. Stained controls 
were compared with control slides stained prior to the instigation of the 
on-board bake procedure. The historic procedure involved heating sections at 
63-65oC for 35 minutes in a large fan-forced dry-air oven (7).

BCL-2   Mum-1   CD31
BCL-6   Calretinin  SATB2
BOB-1   S100CyclinD1
CD20ALK-1   MPO
CD21Ki67INI-1
CD3 SynaptophsinBRG-1
HMB-45  ChromograninInhibin
Melan A MyogeninDesmin

The results showed that there was no difference between controls stained with 
the historic compared to the on-board baking procedure except for BCL-6 which 
the new procedure gave stronger staining. (see figure).
In conclusion, we expect that on-board baking of sections should allow 
laboratories to have better control over the pre-analytical variables that can 
adversely affect the immunohistochemistry staining.

References
1.  Henwood, A. F. (2005). Effect of slide drying at 80oC on 
immunohistochemistry. Journal of Histotechnology, 28(1), 45-46.
2.  Wakins, J., Kellock, D., Gillet, C., Egan, M., Pontin, J. E., Millis, 
R. R., & Levinson, D. A. (1990). Enhancement of immunostaining. Histopathology, 
17(2), 185-185.
3.  Dodson, A., Davies, E., & Waring, J. (1991). APTES, a section adhesive 
for immunocytochemistry; and experiences of slide drying at room temperature. 
Histopathology, 19(5), 484-485.
4.  Oates J. (1993) The effect of temperature on immunostaining. Br J 
Biomed Sci 50: 157-158,
5.  Williams, J. H., Mepham, B. L., & Wright, D. H. (1997). Tissue 
preparation for immunocytochemistry. Journal of clinical pathology, 50(5), 
422-428.
6.  Jones, W. T., Stockard, C. R., & Grizzle, W. E. (2001). Effects of time 
and temperature during attachment of sections to microscope slides on 
immunohistochemical detection of antigens. Biotechnic & Histochemistry, 76(2), 
55-58.
7.  Henwood, A. F. (2012). The application of heated detergent 

[Histonet] Unstained slides precut for IHC

[Carrie Disbrow via Histonet]

Hi! Current lab is cutting extra slides for IHC and putting in 60 degree oven 
for 45 mins to dry.  If IHC is ordered a precut control and the already baked 
slides are again put in the 60 degree oven for 45 mins. Sometimes the control 
is cut and added to the dry slide or a separate slide.  So, the questions  have 
been should the   slides be air dried first using a fan before baking and 
slides not baked until the pathologist places an order? Should the tissue and 
the control all have the same amount of time in the oven to ensure consistency? 
Also, is it better to rack slides standing or on edge for IHC? Additionally, 
when sending slides for IHC to other labs is it preferred to send dry slides or 
baked slides? Thanks for your input!
Carrie Disbrow, HTL

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