I apologise for the delay in responding to you, I have at last received feedback from the laboratory that tested ab33483 and include their response below:
I have had a look through your customers protocol and note that it differs somewhat from our recommended protocol for cryostat sections.
So firstly from their protocols:
1. I note sections appear to be stored in a freezer prior to staining. Some antigens are more sensitive to this than others, this may be the case here. I would recommend using freshly sectioned material
2. Following acetone fixation they have included a triton step (for the DAPI?). This may not be compatible with staining for CD11c using this antibody. I would suggest omitting this step. I note from the literature that such a step is not included in publications reporting successful staining with this antibody.
3. We do not support the use of Immunofluorescence for this antibody. I do not know whether there is sufficient sensitivity to allow the use of TRIT-C for its detection. I would suggest the use of a peroxidase-DAB protocol as has been used in all the publication reporting successful.
My gut feeling is that it may be the Triton X-100 that may affect the epitope recognised by the antibody. Just omitting this step may help restore antigenicity.
Please find a list of publications where this has been used successfully for IHC on cryostat sections. There may be further appropriate advice in these.
1: Witmer-Pack MD, Crowley MT, Inaba K, Steinman RM.
Macrophages, but not dendritic cells, accumulate colloidal carbon following
administration in situ.
J Cell Sci. 1993 Aug;105 ( Pt 4):965-73.
2: Metlay JP, Witmer-Pack MD, Agger R, Crowley MT, Lawless D, Steinman RM.
The distinct leukocyte integrins of mouse spleen dendritic cells as identified
with new hamster monoclonal antibodies.
J Exp Med. 1990 May 1;171(5):1753-71.
3: McMenamin PG.
Dendritic cells and macrophages in the uveal tract of the normal mouse eye.
Br J Ophthalmol. 1999 May;83(5):598-604.
4: Pimorady-Esfahani A, Grounds MD, McMenamin PG.
Macrophages and dendritic cells in normal and regenerating murine skeletal
Muscle Nerve. 1997 Feb;20(2):158-66.
5: Kelsall BL, Strober W.
Distinct populations of dendritic cells are present in the subepithelial dome
and T cell regions of the murine Peyer's patch.
J Exp Med. 1996 Jan 1;183(1):237-47.
6: Dahlen E, Dawe K, Ohlsson L, Hedlund G.
Dendritic cells and macrophages are the first and major producers of TNF-alpha
in pancreatic islets in the nonobese diabetic mouse.
J Immunol. 1998 Apr 1;160(7):3585-93.
7: Leenen PJ, Radosevic K, Voerman JS, Salomon B, van Rooijen N, Klatzmann D,
van Ewijk W.
Heterogeneity of mouse spleen dendritic cells: in vivo phagocytic activity,
expression of macrophage markers, and subpopulation turnover.
J Immunol. 1998 Mar 1;160(5):2166-73.
8: Gonzalez-Juarrero M, Orme IM.
Characterization of murine lung dendritic cells infected with Mycobacterium
Infect Immun. 2001 Feb;69(2):1127-33.
9: Hamada H, Hiroi T, Nishiyama Y, Takahashi H, Masunaga Y, Hachimura S,
Kaminogawa S, Takahashi-Iwanaga H, Iwanaga T, Kiyono H, Yamamoto H, Ishikawa H.
Identification of multiple isolated lymphoid follicles on the antimesenteric
wall of the mouse small intestine.
J Immunol. 2002 Jan 1;168(1):57-64.
10: Bjorck P.
Dendritic cells exposed to herpes simplex virus in vivo do not produce
IFN-alpha after rechallenge with virus in vitro and exhibit decreased T cell
J Immunol. 2004 May 1;172(9):5396-404.
Please note that most of these appear to be available freely via PubMed. Reference 1 has some great pictures of staining in the liver and in spleen with N418 and other antibodies such as F4/80 and FA-11 etc.
I hope this advice will be useful. Please do not hesitate to contact me if you still experience problems with those recommendations, if you purchased the antibody in the last 90 days I would be happy to offer you a refund,